Grp78-binding antibodies and uses thereof and selection of phage-displayed accessible recombinant targeted antibodies

ABSTRACT

Isolated or recombinant EphA5 or GRP78 targeting antibodies are provided. In some cases, antibodies of the embodiments can be used for the detection, diagnosis and/or therapeutic treatment of human diseases, such as cancer. A method of rapidly identifying antibodies or antibody fragments for the treatment of cancer using a combination of in vitro and in vivo methodologies is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 17/592,828, filed Feb. 4, 2022, which is adivisional of, and claims priority to, U.S. application Ser. No.16/333,898, filed Mar. 15, 2019, now issued as U.S. Pat. No. 11,254,751,which is a 35 U.S.C. § 371 national phase application from, and claimspriority to, PCT International Application No. PCT/US2017/052661, filedSep. 21, 2017, which claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 62/397,512, filed Sep. 21,2016, and 62/397,521, filed Sep. 21, 2016, each of said applicationsbeing incorporated by reference in their entireties herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under grant numberDK093500 awarded by the National Institutes of Health and grant numberDE-AC52-06NA25396 awarded by the Department of Energy. The Governmenthas certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM

This invention contains one or more sequences in a computer readableformat in an accompanying text file titled“370602-7050US3_Sequence_Listing,” which is 40.0 KB in size and wascreated May 29, 2022, the contents of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the field of cancer biology.More particularly, it concerns targeting antibodies for the treatmentand detection of cancer. In additional embodiments,

Description of Related Art

The development of targeted tools for cancer therapy is a major focus inoncology. Monoclonal antibody based therapies can have promisingbeneficial results, but standard approaches to generate antibodiesagainst targets of interest with therapeutic properties are timeconsuming and not always successful.

The concept of targeted cancer therapy is predicated on the assumptionthat tumors have unique and sustained genetic abnormalities, and thatdirect targeting of such distinctive biological features can only beaccomplished through the identification and validation of certainspecific molecular markers. These principles are particularly relevantto lung cancer, the leading cause of cancer-related death worldwide(Ferlay et al., 2010, Int J Cancer 127: 2893-2917; Jemal et al., 2010,Cancer epidemiology, biomarkers & prevention 19:1893-1907; Jemal et al,2010, Cancer J Clin 60:277-300). The discovery of novel targets and thedevelopment of therapies and diagnostics (such as those based onmonoclonal antibodies) promise to revolutionize anti-cancer therapy andcancer cell imaging techniques. However, there remains and urgent unmetneed to identify cancer cell binding agents, such as antibodies thatbind to EphA5, that are effective for therapy and/or imaging.

Ephrin type-A receptor 5 is a protein that is encoded by the EphA5 genein humans. Ephrin receptors belong to a family of closely relatedproteins with diverse functions in both normal physiology and diseasepathogenesis (Pasquale 2008; Pasquale 2010). EphA5 is mostly recognizedfor its critical role in axonal guidance during embryonic development(Taylor et al. 1994; Zhou 1997; Murai and Pasquale 2002); itsinvolvement in cancer is only recently becoming evident (Fu et al. 2010;Giaginis et al. 2010; Pejovic et al. 2009).

GRP78, also known as heat shock protein-5 (Hspa5/BiP), is part of anevolutionarily conserved, ER-linked stress response mechanism thatprovides cellular survival signals during environmental and physiologicduress. As a molecular component of the ER chaperoning network, GRP78 isclassically involved with the processing of unfolded proteins, howevernewer insights also place the protein at the cell surface with thepotential to influence signal transduction (Lee, 2014, Nature Rev Cancer14(4): 263-276). Retrospective IHC studies have demonstrated that GRP78expression positively correlates with poor survival in advanced breastcancer (Lee et al., 2006, Cancer Research 66(16): 7849-7853) andrecurrence in prostate cancer patients (Daneshmand et al., 2007, HumanPathology 38(10): 1547-1552). Upregulation of GRP78 promotes survivaland chemoresistance in both proliferating and dormant breast cancercells. Synthetic peptides composed of GRP78-binding motifs coupled to acell death-inducing peptide to promote apoptosis in cancer cells,demonstrating its in vivo accessibility (Arap et al., 2004, Cancer Cell6(3): 275-284).

Monoclonal antibody-based therapy of human cancers has emerged as amajor advance in contemporary medical oncology. However, theidentification of suitable cancer target candidates is merely theinitial step towards the development of clinical applications.Cancer-specific targets are often abundantly present on the surface ofcancer cells or non-malignant tumor-associated vascular endothelial andstromal cells, and are thereby accessible from the systemic circulation(Ozawa et al., 2008). Such targets usually consist of a broad array ofproteins that are overexpressed, mutated, or abnormally located in thecell surface compared to normal tissues (Carter et al., 2004).Conventionally, once a target candidate is identified and validated,panels of antibodies are produced, and evaluated for biological activityand favorable immune profiles prior to drug lead optimization. The morerecent use of in vitro technologies, such as phage- and yeast-display,to generate monoclonal antibody clones has some advantages overtraditional immunization. These include the speed with which antibodyclones can be selected and isolated, the ability to enrich for specificproperties in high-throughput and, perhaps most importantly, the factthat human monoclonal antibodies can be selected directly.

SUMMARY OF THE INVENTION EphA5 Binding Monoclonal Antibodies

Described herein are EphA5 monoclonal antibodies or GRP78 monoclonalantibodies that potently block or reduce EphA5 signaling and GRP78signaling and inhibit cancer cell proliferation. Thus, in a firstembodiment, there is provided an isolated or recombinant monoclonalantibody that specifically binds to a EphA5. In certain aspects, anantibody that competes for the binding of a EphA5 with the E31, F31,TW3, or T22 antibody is provided. In certain aspects, the antibody maycomprise all or part of the heavy chain variable region and/or lightchain variable region of the E31, F31, TW3, or T22 antibodies. In afurther aspect, the antibody may comprise an amino acid sequence thatcorresponds to a first, second, and/or third complementarity determiningregion (CDR) from the light variable and/or heavy variable chain of theE31, F31, TW3, or T22 antibodies of the present embodiments.

In certain aspects, the isolated EphA5 antibody comprises CDR sequencesat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to the CDR regions of the E31, F31, TW3, or T22 heavy andlight chain amino acid sequences. In further aspects, an antibodycomprises CDR regions identical to E31, F31, TW3, or T22 CDR regions,except for one or two amino acid substitutions, deletions, or insertionsat one or more of the CDRs. For example, the antibody can comprise CDRswherein the CDR sequences comprise 1 or 2 amino acid substitutions inthe V_(H) CDR1, V_(H) CDR2, V_(H) CDR3, V_(L) CDR1, V_(L) CDR2 and/orV_(L) CDR3 relative to the CDRs of a E31, F31, TW3, or T22 antibody.Thus, in some specific aspects, an antibody of the embodiments comprises(a) a first V_(H) CDR at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to V_(H) CDR1 of E31 (SEQ ID NO:3), F31 (SEQ ID NO: 11), TW3 (SEQ ID NO: 19), or T22 (SEQ ID NO: 27);(b) a second V_(H) CDR at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to V_(H) CDR2 of E31 (SEQ ID NO:4), F31 (SEQ ID NO: 12), TW3 (SEQ ID NO: 20), or T22 (SEQ ID NO: 28);(c) a third V_(H) CDR at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to V_(H) CDR3 of E31 (SEQ ID NO:5), F31 (SEQ ID NO: 13), TW3 (SEQ ID NO: 21), or T22 (SEQ ID NO: 29);(d) a first V_(L) CDR at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to V_(L) CDR1 of E31 (SEQ ID NO:6), F31 (SEQ ID NO: 14), TW3 (SEQ ID NO: 22), or T22 (SEQ ID NO: 30);(e) a second V_(L) CDR at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to V_(L) CDR2 of E31 (SEQ ID NO:7), F31 (SEQ ID NO: 15), TW3 (SEQ ID NO: 23), or T22 (SEQ ID NO: 31);and (f) a third V_(L) CDR at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to V_(L) CDR3 of E31 (SEQ IDNO: 8), F31 (SEQ ID NO: 16), TW3 (SEQ ID NO: 24), or T22 (SEQ ID NO:32). In certain aspects, such an antibody is a humanized or de-immunizedantibody comprising the foregoing CDRs on a human IgGs (e.g., IgG1,IgG2, IgG4, or a genetically modified IgG) backbone.

In further aspects, the isolated antibody comprises a first V_(H), asecond V_(H), a third V_(H), a first V_(L), a second V_(L), and a thirdV_(L) CDR sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to the corresponding CDR sequence of theE31 antibody, which are represented by SEQ ID NOs: 3, 4, 5, 6, 7, and 8,respectively. In one aspect, the isolated antibody comprises CDRsequences that are identical to the CDR sequences of E31 antibody.

In still further aspects, the isolated antibody comprises a first V_(H),a second V_(H), a third V_(H), a first V_(L), a second V_(L), and athird V_(L) CDR sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding CDRsequence of the F31 antibody, which are represented by SEQ ID NOs: 11,12, 13, 14, 15, and 16, respectively. In one aspect, the isolatedantibody comprises CDR sequences that are identical to the CDR sequencesof F31 antibody.

In other aspects, the isolated antibody comprises a first V_(H), asecond V_(H), a third V_(H), a first V_(L), a second V_(L), and a thirdV_(L) CDR sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to the corresponding CDR sequence of theTW3 antibody, which are represented by SEQ ID NOs: 19, 20, 21, 22, 23,and 24, respectively. In one aspect, the isolated antibody comprises CDRsequences that are identical to the CDR sequences of TW3 antibody.

In yet still further aspects, the isolated antibody comprises a firstV_(H), a second V_(H), a third V_(H), a first V_(L), a second V_(L), anda third V_(L) CDR sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding CDRsequence of the T22 antibody, which are represented by SEQ ID NOs: 27,28, 29, 30, 31, and 32, respectively. In one aspect, the isolatedantibody comprises CDR sequences that are identical to the CDR sequencesof T22 antibody.

In another aspect, the isolated antibody comprises a V_(H) domain atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% identical to the V_(H) domain of E31 (SEQ ID NO: 1), F31 (SEQ IDNO: 9), TW3 (SEQ ID NO: 17), or T22 (SEQ ID NO: 25); and a V_(L) domainat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identical to the V_(L) domain of E31 (SEQ ID NO: 2), F31(SEQ ID NO: 10), TW3 (SEQ ID NO: 18), or T22 (SEQ ID NO: 26). Forexample, the antibody can comprise a V_(H) domain at least 95% identicalto the V_(H) domain of the humanized E31 antibody and a V_(L) domain atleast 95% identical to the V_(L) domain of the humanized E31 antibody.Thus, in some aspects, an antibody comprises a V_(H) domain identical tothe V_(H) domain of humanized E31 antibody and a V_(L) domain identicalto the V_(L) domain of the humanized E31 antibody. In a specificexample, the isolated antibody can comprise V_(H) and V_(L) domainsidentical to those of the E31 antibody. In other aspects, the antibodycomprises a V_(H) domain at least 95% identical to the V_(H) domain ofthe humanized F31 antibody clone and a V_(L) domain at least 95%identical to the V_(L) domain of the humanized F31 antibody. Forinstance the antibody can comprise a V_(H) domain identical to the V_(H)domain of the humanized F31 antibody and a V_(L) domain identical to theV_(L) domain of the humanized F31 antibody. In another specific example,the isolated antibody can comprise V_(H) and V_(L) domains identical tothose of the F31 antibody. In still further aspects, the antibodycomprises a V_(H) domain at least 95% identical to the V_(H) domain ofthe humanized TW3 antibody and a V_(L) domain at least 95% identical tothe V_(L) domain of the humanized TW3 antibody. For instance theantibody can comprise a V_(H) domain identical to the V_(H) domain ofthe humanized TW3 antibody and a V_(L) domain identical to the V_(L)domain of the humanized TW3 antibody. In a particular example, theisolated antibody can comprise V_(H) and V_(L) domains identical tothose of the TW3 antibody. In another aspect, the antibody comprises aV_(H) domain at least 95% identical to the V_(H) domain of the humanizedT22 antibody and a V_(L) domain at least 95% identical to the V_(L)domain of the humanized T22 antibody. For instance the antibody cancomprise a V_(H) domain identical to the V_(H) domain of the humanizedT22 antibody and a V_(L) domain identical to the V_(L) domain of thehumanized T22 antibody. In a certain aspect, the isolated antibody cancomprise V_(H) and V_(L) domains identical to those of the T22 antibody.

In some aspects, an antibody of the embodiments may be an IgG (e.g.,IgG1, IgG2, IgG3 or IgG4), IgM, IgA, genetically modified IgG isotype,or an antigen binding fragment thereof. The antibody may be a Fab′, aF(ab′)2 a F(ab′)3, a monovalent scFv, a bivalent scFv, a bispecific or asingle domain antibody. The antibody may be a human, humanized, orde-immunized antibody. In a further aspect, the isolated antibody is theE31, F31, TW3, or T22 antibody.

In some aspects, the antibody may be conjugated to an imaging agent, achemotherapeutic agent, a toxin, or a radionuclide. In specific aspects,the antibody may be conjugated to auristatin or to monomethyl auristatinE (MMAE) in particular.

In one embodiment, there is provided a recombinant polypeptidecomprising an antibody V_(H) domain comprising CDRs 1-3 of the V_(H)domain of E31 (SEQ ID NOs: 3, 4, and 5), F31 (SEQ ID NOs: 11, 12, and13), TW3 (SEQ ID NOs: 19, 20, and 21), or T22 (SEQ ID NOs: 27, 28, and29). In another embodiment, there is provided a recombinant polypeptidecomprising an antibody V_(L) domain comprising CDRs 1-3 of the V_(L)domain of E31 (SEQ ID NOs: 6, 7, and 8), F31 (SEQ ID NOs: 14, 15, and16), TW3 (SEQ ID NOs: 22, 23, and 24), or T22 (SEQ ID NOs: 30, 31, and32).

In some embodiments, there is provided an isolated polynucleotidemolecule comprising nucleic acid sequence encoding an antibody or apolypeptide comprising an antibody V_(H) or V_(L) domain disclosedherein.

In further embodiments, a host cell is provided that produces amonoclonal antibody or recombinant polypeptide of the embodiments. Insome aspects, the host cell is a mammalian cell, a yeast cell, abacterial cell, a ciliate cell, or an insect cell. In certain aspects,the host cell is a hybridoma cell.

In still further embodiments, there is provided a method ofmanufacturing an antibody of the present invention comprising expressingone or more polynucleotide molecule(s) encoding a V_(L) or V_(H) chainof an antibody disclosed herein in a cell and purifying the antibodyfrom the cell.

In additional embodiments, there are pharmaceutical compositionscomprising an antibody or antibody fragment as discussed herein. Such acomposition further comprises a pharmaceutically acceptable carrier andmay or may not contain additional active ingredients.

In embodiments of the present invention, there is provided a method fortreating a subject having a cancer comprising administering an effectiveamount of an antibody disclosed herein. In certain aspects, the antibodyis a monoclonal antibody of the embodiments herein, such as the E31,F31, TW3, or T22 antibody or a recombinant polypeptide comprisingantibody segment derived therefrom.

In certain aspects, the cancer may be a breast cancer, lung cancer, head& neck cancer, prostate cancer, esophageal cancer, tracheal cancer,brain cancer, liver cancer, bladder cancer, stomach cancer, pancreaticcancer, ovarian cancer, uterine cancer, cervical cancer, testicularcancer, colon cancer, rectal cancer or skin cancer. In specific aspects,the cancer is an epithelial cancer. In other aspects, cancer may be acolorectal adenocarcinoma, lung adenocarcinoma, lung squamous cellcarcinoma, breast cancer, hepatocellular carcinoma, ovarian cancer,kidney renal clear cell carcinoma, lung cancer or kidney cancer.

In one aspect, the antibody may be administered systemically. Inadditional aspects, the antibody may be administered intravenously,intradermally, intratumorally, intramuscularly, intraperitoneally,subcutaneously, or locally. The method may further compriseadministering at least a second anticancer therapy to the subject.Examples of the second anticancer therapy include, but are not limitedto, surgical therapy, chemotherapy, radiation therapy, cryotherapy,hormonal therapy, immunotherapy, or cytokine therapy.

In further aspects, the method may further comprise administering acomposition of the present invention more than one time to the subject,such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or moretimes.

In another embodiment, there is provided a method for detecting a cancerin a subject comprising testing for the presence of elevated EphA5relative to a control in a sample from the subject, wherein the testingcomprises contacting the sample with an antibody disclosed herein. Forexample, the method may be an in vitro or in vivo method.

Certain embodiments are directed to an antibody or recombinantpolypeptide composition comprising an isolated and/or recombinantantibody or polypeptide that specifically binds EphA5. In certainaspects the antibody or polypeptide has a sequence that is, is at least,or is at most 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical (or anyrange derivable therein) to all or part of any antibody provided herein.In still further aspects the isolated and/ or recombinant antibody orpolypeptide has, has at least, or has at most 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more contiguousamino acids from any of the sequences provided herein or a combinationof such sequences.

In still further aspects, an antibody or polypeptide of the embodimentscomprises one or more amino acid segments of the any of the amino acidsequences disclosed herein. For example, the antibody or polypeptide cancomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid segmentscomprising about, at least or at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, or 120 amino acids in length, including all valuesand ranges there between, that are at least 80, 85, 90, 95, 96, 97, 98,99, or 100% identical to any of the amino acid sequences disclosedherein. In certain aspects the amino segment(s) are selected from one ofthe amino acid sequences of a EphA5-binding antibody as provided herein.

In still further aspects, an antibody or polypeptide of the embodimentscomprises an amino acid segment of the any of the amino acid sequencesdisclosed herein, wherein the segment begins at amino acid position 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 inany sequence provided herein and ends at amino acid position 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, or 120 in the same providedsequence. In certain aspects the amino segment(s), or portions thereof,are selected from one of the amino acid sequences of a EphA5-bindingantibody as provided herein.

In yet further aspects, an antibody or polypeptide of the embodimentscomprises an amino acid segment that is at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any rangederivable therein) to a V, VJ, VDJ, D, DJ, J or CDR domain of aEphA5-binding antibody (as provided in Table 1). For example, apolypeptide may comprise 1, 2 or 3 amino acid segment that are at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical (or any range derivable therein) to CDRs 1, 2, and/or 3 aEphA5-binding antibody as provided in Table 1.

GRP78 Binding Monoclonal Antibodies

Embodiments of the present disclosure provide antibodies and methods oftreating cancer employing such antibodies. In some embodiments, thereare provided GRP78 specific human monoclonal antibodies. A unique methodof combining phage and yeast display selection in vitro with tumorxenograft selection in vivo is provided. This method identifies highlyspecific and tumor cell-localizing single chain antibodies, asexemplified herein. The present disclosure also provides therapeuticcompositions comprising GRP78 specific antibodies or antigen bindingfragments thereof In preferred applications, fully human GRP78antibodies and antigen binding fragments thereof are provided andemployed in the generation of antibody-drug conjugates suitable fortherapeutic administration to cancer patients. Without limitation, GRP78positive breast and prostate cancer patients are expected to beexcellent candidates for therapy with the compositions and methods ofthe invention.

Described herein are monoclonal antibodies against GRP78. In furtheraspects, provided GRP78-binding antibodies reduce GRP78 signaling andcan be used to inhibit cancer cell proliferation. Thus, in a firstembodiment, there is provided an isolated or recombinant monoclonalantibody that specifically binds to a GRP78. In certain aspects, anantibody that competes for the binding of a GRP78 with the B4, D1, or F6antibody is provided. In certain aspects, the antibody may comprise allor part of the heavy chain variable region and/or light chain variableregion of the B4, D1, or F6 antibodies. In a further aspect, theantibody may comprise an amino acid sequence that corresponds to afirst, second, and/or third complementarity determining region (CDR)from the light variable and/or heavy variable chain of the B4, D1, or F6antibodies of the present embodiments.

In certain aspects, the isolated antibody comprises CDR sequences atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the CDR regions of the B4, D1, or F6 heavy and light chainamino acid sequences. In further aspects, an antibody comprises CDRregions identical to the B4, D1, or F6 CDR regions, except for one ortwo amino acid substitutions, deletions, or insertions at one or more ofthe CDRs. For example, the antibody can comprise CDRs wherein the CDRsequences comprise 1 or 2 amino acid substitutions in the V_(H) CDR1,V_(H) CDR2, V_(H) CDR3, V_(L) CDR1, V_(L) CDR2 and/or V_(L) CDR3relative to the CDRs of a B4, D1, or F6 antibody. Thus, in some specificaspects, an antibody of the embodiments comprises (a) a first V_(H) CDRat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to V_(H) CDR1 of B4 (SEQ ID NO: 39), D1 (SEQ ID NO: 47),or F6 (SEQ ID NO: 55); (b) a second V_(H) CDR at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to V_(H)CDR2 of B4 (SEQ ID NO: 40), D1 (SEQ ID NO: 48), or F6 (SEQ ID NO: 56);(c) a third V_(H) CDR at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to V_(H) CDR3 of B4 (SEQ ID NO:41), D1 (SEQ ID NO: 49), or F6 (SEQ ID NO: 57); (d) a first V_(L) CDR atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to V_(L) CDR1 of B4 (SEQ ID NO: 42), D1 (SEQ ID NO: 50), or F6(SEQ ID NO: 58); (e) a second V_(L) CDR at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to V_(L) CDR2of B4 (SEQ ID NO: 43), D1 (SEQ ID NO: 51), or F6 (SEQ ID NO: 59); and(f) a third V_(L) CDR at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to V_(L) CDR3 of B4 (SEQ ID NO:44), D1 (SEQ ID NO: 52), or F6 (SEQ ID NO: 60). In certain aspects, suchan antibody is a humanized or de-immunized antibody comprising theforegoing CDRs on a human IgGs (e.g., IgG1, IgG2, IgG4, or a geneticallymodified IgG) backbone.

In further aspects, the isolated antibody comprises a first V_(H), asecond V_(H), a third V_(H), a first V_(L), a second V_(L), and a thirdV_(L) CDR sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to the corresponding CDR sequence of theB4 antibody, which are represented by SEQ ID NOs: 3, 4, 5, 6, 7, and 8,respectively. In one aspect, the isolated antibody comprises CDRsequences that are identical to the CDR sequences of B4 antibody.

In still further aspects, the isolated antibody comprises a first V_(H),a second V_(H), a third V_(H), a first V_(L), a second V_(L), and athird V_(L) CDR sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding CDRsequence of the D1 antibody, which are represented by SEQ ID NOs: 11,12, 13, 14, 15, and 16, respectively. In one aspect, the isolatedantibody comprises CDR sequences that are identical to the CDR sequencesof D1 antibody.

In other aspects, the isolated antibody comprises a first V_(H), asecond V_(H), a third V_(H), a first V_(L), a second V_(L), and a thirdV_(L) CDR sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to the corresponding CDR sequence of theF6 antibody, which are represented by SEQ ID NOs: 19, 20, 21, 22, 23,and 24, respectively. In one aspect, the isolated antibody comprises CDRsequences that are identical to the CDR sequences of F6 antibody.

In another aspect, the isolated antibody comprises a V_(H) domain atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% identical to the V_(H) domain of B4 (SEQ ID NO: 1), D1 (SEQ IDNO: 9), or F6 (SEQ ID NO: 17); and a V_(L) domain at least about 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the V_(L) domain of B4 (SEQ ID NO: 2), D1 (SEQ ID NO: 10), or F6 (SEQID NO: 18). For example, the antibody can comprise a V_(H) domain atleast 95% identical to the V_(H) domain of the humanized B4 antibody anda V_(L) domain at least 95% identical to the V_(L) domain of thehumanized B4 antibody. Thus, in some aspects, an antibody comprises aV_(H) domain identical to the V_(H) domain of humanized B4 antibody anda V_(L) domain identical to the V_(L) domain of the humanized B4antibody. In a specific example, the isolated antibody can compriseV_(H) and V_(L) domains identical to those of the B4 antibody. In otheraspects, the antibody comprises a V_(H) domain at least 95% identical tothe V_(H) domain of the humanized D1 antibody and a V_(L) domain atleast 95% identical to the V_(L) domain of the humanized D1 antibody.For instance the antibody can comprise a V_(H) domain identical to theV_(H) domain of the humanized D1 antibody and a V_(L) domain identicalto the V_(L) domain of the humanized D1 antibody. In another specificexample, the isolated antibody can comprise V_(H) and V_(L) domainsidentical to those of the D1 antibody. In still further aspects, theantibody comprises a V_(H) domain at least 95% identical to the V_(H)domain of the humanized F6 antibody and a V_(L) domain at least 95%identical to the V_(L) domain of the humanized F6 antibody. For instancethe antibody can comprise a V_(H) domain identical to the V_(H) domainof the humanized F6 antibody and a V_(L) domain identical to the V_(L)domain of the humanized F6 antibody. In a particular example, theisolated antibody can comprise V_(H) and V_(L) domains identical tothose of the F6 antibody.

In some aspects, an antibody of the embodiments may be an IgG (e.g.,IgG1, IgG2, IgG3 or IgG4), IgM, IgA, genetically modified IgG isotype,or an antigen binding fragment thereof. The antibody may be a Fab′, aF(ab′)2 a F(ab′)3, a monovalent scFv, a bivalent scFv, a bispecific or asingle domain antibody. The antibody may be a human, humanized, orde-immunized antibody. In a further aspect, the isolated antibody is theB4, D1, or F6 antibody.

In some aspects, the antibody may be conjugated to an imaging agent, achemotherapeutic agent, a toxin, or a radionuclide. In specific aspects,the antibody may be conjugated to auristatin or to monomethyl auristatinE (MMAE) in particular.

In one embodiment, there is provided a recombinant polypeptidecomprising an antibody V_(H) domain comprising CDRs 1-3 of the V_(H)domain of B4 (SEQ ID NOs: 3, 4, and 5), D1 (SEQ ID NOs: 11, 12, and 13),or F6 (SEQ ID NOs: 19, 20, and 21). In another embodiment, there isprovided a recombinant polypeptide comprising an antibody V_(L) domaincomprising CDRs 1-3 of the V_(L) domain of B4 (SEQ ID NOs: 6, 7, and 8),D1 (SEQ ID NOs: 14, 15, and 16), or F6 (SEQ ID NOs: 22, 23, and 24).

In some embodiments, there is provided an isolated polynucleotidemolecule comprising nucleic acid sequence encoding an antibody or apolypeptide comprising an antibody V_(H) or V_(L) domain disclosedherein.

In further embodiments, a host cell is provided that produces amonoclonal antibody or recombinant polypeptide of the embodiments. Insome aspects, the host cell is a mammalian cell, a yeast cell, abacterial cell, a ciliate cell, or an insect cell. In certain aspects,the host cell is a hybridoma cell.

In still further embodiments, there is provided a method ofmanufacturing an antibody of the present invention comprising expressingone or more polynucleotide molecule(s) encoding a V_(L) or V_(H) chainof an antibody disclosed herein in a cell and purifying the antibodyfrom the cell.

In additional embodiments, there are pharmaceutical compositionscomprising an antibody or antibody fragment as discussed herein. Such acomposition further comprises a pharmaceutically acceptable carrier andmay or may not contain additional active ingredients.

In embodiments of the present invention, there is provided a method fortreating a subject having a cancer comprising administering an effectiveamount of an antibody disclosed herein. In certain aspects, the antibodyis a monoclonal antibody of the embodiments herein, such as the B4, D1,or F6 antibody or a recombinant polypeptide comprising antibody segmentderived therefrom.

In certain aspects, the cancer may be a breast cancer, lung cancer, head& neck cancer, prostate cancer, esophageal cancer, tracheal cancer,brain cancer, liver cancer, bladder cancer, stomach cancer, pancreaticcancer, ovarian cancer, uterine cancer, cervical cancer, testicularcancer, colon cancer, rectal cancer or skin cancer. In specific aspects,the cancer is an epithelial cancer. In other aspects, cancer may be acolorectal adenocarcinoma, lung adenocarcinoma, lung squamous cellcarcinoma, breast cancer, hepatocellular carcinoma, ovarian cancer,kidney renal clear cell carcinoma, lung cancer or kidney cancer.

In one aspect, the antibody may be administered systemically. Inadditional aspects, the antibody may be administered intravenously,intradermally, intratumorally, intramuscularly, intraperitoneally,subcutaneously, or locally. The method may further compriseadministering at least a second anticancer therapy to the subject.Examples of the second anticancer therapy include, but are not limitedto, surgical therapy, chemotherapy, radiation therapy, cryotherapy,hormonal therapy, immunotherapy, or cytokine therapy.

In further aspects, the method may further comprise administering acomposition of the present invention more than one time to the subject,such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or moretimes.

In another embodiment, there is provided a method for detecting a cancerin a subject comprising testing for the presence of elevated GRP78relative to a control in a sample from the subject, wherein the testingcomprises contacting the sample with an antibody disclosed herein. Forexample, the method may be an in vitro or in vivo method.

Certain embodiments are directed to an antibody or recombinantpolypeptide composition comprising an isolated and/or recombinantantibody or polypeptide that specifically binds GRP78. In certainaspects the antibody or polypeptide has a sequence that is, is at least,or is at most 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical (or anyrange derivable therein) to all or part of any antibody provided herein.In still further aspects the isolated and/ or recombinant antibody orpolypeptide has, has at least, or has at most 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more contiguousamino acids from any of the sequences provided herein or a combinationof such sequences.

In still further aspects, an antibody or polypeptide of the embodimentscomprises one or more amino acid segments of the any of the amino acidsequences disclosed herein. For example, the antibody or polypeptide cancomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid segmentscomprising about, at least or at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, or 120 amino acids in length, including all valuesand ranges there between, that are at least 80, 85, 90, 95, 96, 97, 98,99, or 100% identical to any of the amino acid sequences disclosedherein. In certain aspects the amino segment(s) are selected from one ofthe amino acid sequences of a GRP78-binding antibody as provided herein.

In still further aspects, an antibody or polypeptide of the embodimentscomprises an amino acid segment of the any of the amino acid sequencesdisclosed herein, wherein the segment begins at amino acid position 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 inany sequence provided herein and ends at amino acid position 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, or 120 in the same providedsequence. In certain aspects the amino segment(s), or portions thereof,are selected from one of the amino acid sequences of a GRP78-bindingantibody as provided herein.

In yet further aspects, an antibody or polypeptide of the embodimentscomprises an amino acid segment that is at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any rangederivable therein) to a V, VJ, VDJ, D, DJ, J or CDR domain of aGRP78-binding antibody (as provided in Table 1A). For example, apolypeptide may comprise 1, 2 or 3 amino acid segment that are at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical (or any range derivable therein) to CDRs 1, 2, and/or 3 aGRP78-binding antibody as provided in Table 1A.

Selection of Phage-Displayed Accessible Recombinant Targeted Antibodies

Embodiments of the present disclosure relate to a novel and robustantibody discovery methodology, termed Selection of Phage-DisplayAccessible Recombinant Targeted Antibodies (SPARTA). The method of thepresent invention combines an in vitro screening step of a naive humanantibody library against known tumor targets, with in vivo selectionsbased upon tumor-homing capabilities of the pre-enriched antibody pool.This approach overcomes several rate-limiting challenges in thegeneration of human antibodies amenable to rapid translation intomedical applications.

To discover target-specific, biologically active antibodies to commonhuman cancers, the inventors conceived of a two-step in tandemmethodology: Selection of Phage-displayed Accessible RecombinantTargeted Antibodies (termed SPARTA). Unlike blind selection approachesin which there is no knowledge of the target, SPARTA starts with apreviously identified tumor cell surface target, against which anenriched pool of recombinant human antibodies is first generated from alarge naive human library using a high throughput combination of phage-and yeast-display (Ferrara et al., 2012). Antibodies from this pool aresubsequently selected directly in vivo for their tumor targetingattributes. The present inventors have pioneered the experimental use ofin vivo peptide phage-display (Arap et al., 2002; Arap et al., 1998;Driessen et al., 2010; Kolonin et al., 2004; Kolonin et al., 2006; Ozawaet al., 2008; Pasqualini and Ruoslahti, 1996; Rajotte et al., 1998;Staquicini et al., 2011a; Staquicini et al., 2010; Staquicini et al.,2011b) and documented extensively that this approach provides exquisiteadvantages in identifying accessible target receptors in the uniquecontext of the native tumor microenvironment. The inventors and otherinvestigators have attempted to extend the in vivo methodology toantibody phage-display libraries, but with only modest success(Deramchia et al., 2012; Krag et al., 2006; Sanchez-Martin et al., 2015;Shukla et al., 2013) likely due to inherent technical constraints suchas the critical need for helper phage “rescue”, which leads to extremelylow antibody display levels. Moreover, the presence of truncatedantibodies and associated exposure of hydrophobic interfaces generallyincrease non-specific binding to nearly prohibitive background levels.

In the present application, the inventors have focused their efforts ontwo established cancer cell surface targets, Ephrin A5 (EphA5), amolecular target in human lung cancer (Staquicini et al., 2015), and 78kDa glucose-regulated protein (GRP78), a relatively promiscuous targeton the tumor cell surface of several human cancers (Arap et al., 2004;Dobroff et al., 2016; Ferrara et al., 2016). Generation of an enrichedpool of human recombinant antibodies screened against EphA5 and GRP78 invitro, along with the functional selection in vivo of the monoclonalantibody pools in preclinical models of human lung cancer and breastcancer (respectively) enabled the identification of single monoclonalantibody clones with favorable tumor-targeting properties. Theindividual monoclonal antibody clones against EphA5 and GRP78consistently recognized and localized to their cognate tumor targets invivo, and showed effective killing activity as ADCs. The inventorsconclude that SPARTA is a broadly enabling platform for the genericselection of tumor-specific antibodies from large human phage-displayantibody libraries, and it may therefore become a method-of-choice toselect monoclonal antibodies against human cancers, and perhaps alsoagainst certain non-malignant diseases.

Embodiments according relate to a method for selecting phage displayedaccessible recombinant targeted antibodies (SPARTA) comprising the stepsof:

-   1. Serially screening a bacteriophage human recombinant antibody    library comprising about 10¹⁰ to 10¹³, often about 10¹¹ transducing    units (“TU”), in vitro against immobilized recombinant antigens to    provide a phage output pool of approximately 10⁵-10⁷, more often    approximately 10⁶ TU;-   2. Transferring genetic material from the phage output pool to a    yeast display vector and serially screening the modified yeast    display vector against the immobilized recombinant antigens from 1-5    times (often at least twice) and optionally against orthologous    recombinant antigens to eliminate cross reactivity, to provide a    yeast output pool containing from 10³-10⁵, often about 10⁴ TU;-   3. Displaying the yeast output pool in phage (preferably phagemid)    particles, in either mono- or multivalent format; 4. Administering    the phage particles into a tumor bearing laboratory test animal and    allowing-   the phage particles sufficient time to bind to the tumor, wherein    the tumor expresses or likely expresses the antigen to which    antibodies displayed in the yeast output pool bind;-   5. Recovering tumor tissue from the laboratory test animal,    recovering the antibody genes within the phage, e.g. by amplifying    the genetic material obtained from the phage particles which bind to    the tumor tissue, or by infection, and repeating steps 3-5 at least    one additional time (e.g. 1, 2, 3, 4, 5 or 6 times).-   6. Determining clonal diversity and ranking of individual components    of the display vector for identification of lead antibody candidates    for monoclonal antibodies; and-   7. Optionally obtaining and sequencing the genetic material from    phages which express the lead candidate proteins or peptides,    introducing the obtained genetic material into an expression vector    and introducing the expression vector into a cell for expression of    the proteins or peptides.

Embodiments according to the present invention relate to a method forselecting phage displayed accessible recombinant targeted antibodies(SPARTA) comprising the steps:

-   1. Serial screening a bacteriophage human recombinant antibody    library comprising about 10¹⁰ to 10¹³, often about 10¹¹ transducing    units (“TU”), in vitro against immobilized recombinant antigens    (“the screened antigen”) to provide a phage output pool of    approximately 10⁵-10⁷, more often approximately 10⁶ TU;-   2. Transferring genetic material from the phage output pool to a    yeast display vector and serial screening the modified yeast display    vector against the immobilized recombinant antigens from 1-5 times    (often at least twice) to provide a yeast output pool containing    from 10³-10⁵, often about 10⁴ TU;-   3. Optionally and preferably negatively screening the yeast output    pool from step 2 from at least one positive screen conducted and    preferably from each of the positive screens conducted against    orthologous recombinant antigens to minimize cross reactivity with    the orthologous antigens;-   4. Cloning the genetic material from the yeast output pool and    displaying the genetic material in single or multivalent display    vector format in phage (preferably phagemid) particles;-   5. Administering the phage particles into a tumor bearing laboratory    test animal and allowing the phage particles sufficient time to    localize and bind to the tumor, wherein the tumor expresses or    likely expresses the antigen to which antibodies displayed in the    yeast output pool bind;-   6. Recovering tumor tissue from the laboratory test animal,    recovering the antibody genes within the phage by amplifying the    genetic material obtained from the phage particles which bind to the    tumor tissue or by infection and repeating steps 3-5 at least one    additional time (e.g. 1, 2, 3, 4, 5, 6 or more times);-   7. Determining clonal diversity and ranking of individual components    of the display vector for identification of lead antibody candidates    for monoclonal antibodies;-   8. Obtaining and sequencing the genetic material from phages which    express the lead candidate proteins or peptides, introducing the    obtained genetic material into an expression vector and introducing    the expression vector into a cell for expression of the proteins or    peptides.

In preferred embodiments of the general method, the phage libraryinitially screened and the subsequent vectors which are screened expressscFv, Fab, Fab′, F(ab′)2, Fd, Fv, dAb, or isolated CDR antibodyfragments, often scFv or Fab fragments, most preferably scFv fragments.In preferred embodiments the initial (naive) bacteriophage antibodylibrary comprises about 10¹¹ TU and the phage output pool after serialscreening comprises about 10⁶ TU. In preferred embodiments, thebacteriophage is screened from 2-7 times (2, 3, 4, 5, 6 or 7, preferably3-5 times, most often 3 times). In preferred aspects the recombinantantigen is an antigen from a tumor surface protein or polypeptide. Inpreferred aspects, the yeast display vector is screened serially atleast twice, more often twice. In preferred embodiments, the yeastoutput pool after serial screening comprises about 10⁴ TU. In preferredembodiments, the output pools from each serial screening (e.g., theyeast output pool or the phagemid output pool) is negatively screenedagainst orthologous proteins or peptides (often, proteins or peptidesfrom the same family of proteins as the recombinant antigen to which thelibraries are screened) to minimize or eliminate cross-reactivity. Inpreferred embodiments, the genetic material from the yeast output poolis cloned into phagemid particles (step 4) which express the geneticmaterial multivalently. In preferred embodiments, the genetic materialfrom the yeast output pool is cloned and incorporated into phagemidparticles which are expressed in cells (preferably, eukaryotic cells andmore preferably E. Coli or other eukaryotic cells carrying a helperplasmid such as a M13cp-dg3, M13K07, R408, VCSM13, R408d3 or KM13).

In preferred embodiments, the phage (preferably phagemid) displayparticles are serially screened three times. In preferred embodiments,after the first two (or more) screens, the genetic material from thephage, preferably a phagemid display is recloned back into the original(umodified) phage display vector, expressed in multivalent format(preferably, the phage display vector is a phagemid display vectorexpressed in eurkaryotic cells containing a helper plasmid and thephage/phagemid display vector is again screened (final screen in theserial screening). After the final screen of the phage/phagemid displayvector, In certain preferred embodiments, the results of a given screen(e.g. a first “homing” screen or a final screen step in a serialscreening), especially a phage/phagemid in vivo screen of tumor tissue,are collected and the number of bound phage particles in the tumor isassessed and/or compared against control (unbound/naive) tumor tissue,preferably using PCR techniques, especially including real-time PCR. Incertain embodiments, the final in vivo screened phage/phagemid particlesare cloned into a phage vector and the cloned phage vector is furthertested in vivo for their ability to home and localize into tumor tissueselectively and compared to a standard (using control phage withoutantibody modification).

It is unexpected that the method according to the present invention willprovide high polyclonality which allows the selection of diversespecific antibodies. Accordingly, the present method can provideparticularly useful monoclonal antibodies or antibody fragments whichcan be used to produce monoclonal antibodies exhibiting unexpectedbinding to tumor tissue as well as favorable homing characteristicsallowing the rapid development of therapeutic monoclonal antibodiesheretofore unknown in the art.

In additional, more specific embodiments, the present invention isdirected to a method for selecting phage displayed accessiblerecombinant targeted antibodies (SPARTA) comprising the steps of

-   1. Immobilizing a target tumor surface antigen (generally a    recombinant target protein or peptide, often a tumor surface    protein) on a binding surface, often the wells of a microtiter plate    to produce an immobilized protein or peptide;-   2. Exposing said protein or peptide on said binding surface to a    naive bacteriophage library comprising a plurality of bacteriophage,    each bacteriophage displaying at least one (preferably a multivalent    single protein) heterologous protein or protein fragment (peptide)    on the viral surface wherein the bacteriophage comprises genetic    material encoding for the heterologous protein or protein fragment;-   3. Allowing the bacteriophage library sufficient time to bind with    the tumor surface protein to produce protein binding bacteriophage;-   4. Isolating the protein binding bacteriophage to provide a    bacteriophage output pool comprising bacteriphage which bind to the    antigen;-   5. Optionally, repeating steps 2-4 at least once serially with the    protein binding bacteriophage pool obtained after each successive    isolation (up to 10 times, often 1, 2, 3, 4, 5 or 6 times) to obtain    a bacteriphage output pool having about 10⁶ TU;-   6. Optionally, growing (amplifying) said protein binding    bacteriphage in said output pool in a bacterial host after step 4 or    after the last isolation pursuant to step 5 to increase the number    of protein binding bacteriophage from said output pool;-   7. Obtaining and sequencing the DNA within said protein binding    bacteriophage to identify the interacting heterologous proteins or    protein fragments;-   8. Cloning the DNA for the interacting heterologous proteins or    protein fragments obtained from step 7 into a yeast display system    to provide a cloned yeast display system which displays said    interacting heterologous proteins or protein fragments, wherein said    yeast comprises genetic material encoding for the heterologous    proteins or protein fragments;-   9. Exposing said immobilized protein to said cloned yeast display    system and allowing said yeast sufficient time to bind to said    immobilized protein to provide protein binding yeast;-   10. Isolating the protein binding yeast;-   11. Repeating steps 9-10 from 1 to 5 times (e.g. 1, 2, 3, 4 or 5)    with the yeast output obtained after each screening step to provide    a yeast output pool comprising about 10³-10⁵, often about 10⁴ TU;-   12. Obtaining and preferably sequencing the DNA within said protein    binding yeast to identify the interacting heterologous proteins or    protein fragments;-   13. Cloning the DNA for the interacting heterologous proteins or    protein fragments obtained from step 12 into a plasmid vector    (preferably, a phagemid vector having a f1 origin of replication) to    provide a polyclonal phage or phagemid vector pool and    introducing/transforming a bacteria to provide a polyclonal    multivalent phage display pool which displays said interacting    heterologous proteins or protein fragments (in the case of the    phagemid display vector the bacteria used to express the vector    contains a helper plasmid, and is preferably E. coli carrying the    M13cp-dg3 helper plasmid);-   14. Administering said phage display pool obtained from step 13 into    a tumor-bearing laboratory test animal having tumor tissue which    expresses said target tumor surface protein and allowing sufficient    time for said phage/phagemid pool to bind to said target tumor    surface protein in said animal;-   15. Collecting said tumor tissue from said laboratory test animal;-   16. Isolating phage or phagemid particles bound to said tumor    tissue;-   17. Optionally, quantifying the binding of phage or phagemid    particles to tumor tissue (e.g. by quantitative PCR) and comparing    the binding of tumor tissue with a standard (e.g. binding of the    phage particles to non-tumor tissue or to a monoclonal antibody with    known tumor binding specificity);-   18. Obtaining and optionally sequencing the DNA within said phage    particles of step 16 to identify the heterologous proteins or    protein fragments which bind to tumor tissue;-   19. Amplifying the DNA provided in step 18, recloning the DNA into    an unmodified display phage or phagemid display vector and repeating    steps 12-17 at least one additional time (often 1, 2, 3, 4, 5 or 6    times, preferably at least two additional times- a serial    screening);-   20. Isolating the phage or phagemid particles from the final screen    of step 19.-   21. Determining the clonal diversity and ranking of individual    components of the display vector for identification of lead protein    or peptide candidates for monoclonal antibodies.-   22. Obtaining DNA from phage or phagemid particles subjected to step    21 and producing antibodies (preferably antibody fusion proteins    such as scFv-Fc fusions) by cloning the DNA into plasmid vectors,    transfecting eukaryotic cells with said plasmid vectors and growing    said eukaryotic cells; and-   23. Isolating said antibodies from said eukaryotic cells and/or    media in which said eurkaryotic cells are grown.

Embodiments further include an optional step of negatively selecting theantibody pools of steps 4, 5, 6, 10, 12, 16 and/or 19 and/or theantibodies of claim 21 to minimize or eliminate binding to orthologousnon-target antigens.

Embodiments often include a human recombinant tumor surface antigen astarget, a human scFv library as the bacteriophage library (preferablynaive).

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Whole cell phage-ELISA assay to demonstrate specific binding ofanti-EphA5 antibodies to native EphA5 target expressed on H460 humanlung carcinoma cells in vitro. The binding to H460 cells was higher thanH226 in all cases.

FIG. 2. Immunofluorescence cellular imaging, showing intracellularlocation of selected anti-EphA5 scFv antibodies in human carcinomacells. There is moderate staining of E31 and TW3, with stronger stainingof F31 and T22.

FIGS. 3A-3B. Cell viability assay showing that anti-EphA5minibody—secondary antibody-drug conjugates are more effective atkilling human carcinoma cells compared to drug alone.

FIGS. 4A-4D. Secondary Antibody-drug conjugate. Protein G purifiedminibodies were added to cells cultured in 96-wells plates. After a 10minute incubation on ice, secondary antibody-drug conjugates were addedto each well at a final concentration of 20 nM. Cell survival wasassessed in real time for 72 h with the automated xCELLigence System(ACEA Biosciences).

FIG. 5. Tumor targeting of minibodies. Protein G purified minibodieswere injected into mice bearing tumors and allowed to circulate for 6min. After perfusion with PBS and with PFA, tumor tissue and controlswere harvested, embedded and sectioned. Minibody detection in tumor andselected control tissues (liver and pancreas) is shown.

FIGS. 6A-6D. Radiosensitization activity of anti-EphA5 clones. Survivingfractions of H460 cells exposed to increasing doses of IR afteradministration of phage displaying selected anti-EphA5 scFvs. A phagelibrary was used as control. Two out of four tested clones (B and D)showed radiosensitizing properties.

FIG. 7. Phage internalization. Single scFv clones were produced asmonovalent phage and tested for binding and internalization on MCF-7human breast cancer (GRP78 positive) cell line in vitro. An irrelevantscFv displayed on phage was used as negative control. Briefly, the cellswere seeded on microscope slides, blocked and incubated with phage for 4h to allow binding and internalization. After washing, the surface boundphage were stripped, and the cells were fixed and permeabilized. Thephage were detected with a mouse antiM13 mAb (GE) and a Cy3-conjugatedantiMouse secondary Ab (Dako). The slides were mounted with VECTASHIELD®anti-fade mounting medium.

FIG. 8. Phage binding on cells. A whole cell phage-ELISA was used todemonstrate specific binding of the selected anti-GRP78 scFv antibodiesto GRP78 target on a human breast carcinoma cell line in vitro. SinglescFv clones were produced as monovalent phage and tested for binding onMCF-7 in vitro. Briefly, cells were seeded on 96-well plate, blocked andincubated with phage displaying the scFv of interest at differentconcentrations. After washing, cell surface bound phage were detectedwith the antiM13-HRP conjugated mAb (GE). An irrelevant scFv displayedon phage was used as negative control.

FIG. 9. Cell surface binding. Cell surface binding of anti-GRP78minibodies was assessed by flow cytometry on Ef43 murine breast cancercell line. Briefly, Ef43 cells were harvested and incubated for 30 minwith the fluorescently labeled minibodies (antiGRP78, right peak in bothpanels, and isotype control). After extensive washings, the cells wereanalyzed by flow cytometry.

FIGS. 10A-10D. Secondary Antibody-drug conjugate. Protein G purifiedminibodies were added to cells cultured in 96-wells plates. After a 10minute incubation on ice, secondary antibody-drug conjugates were addedto each well at a final concentration of 20 nM. Cell survival wasassessed in real time for 72 h with the automated xCELLigence System(ACEA Biosciences).

FIG. 11. Tumor targeting of minibodies. Protein G purified minibodieswere injected into mice bearing tumors and allowed to circulate for 6min. After perfusion with PBS and with PFA, tumor tissue and controlswere harvested, embedded and sectioned. Minibody detection in tumor andselected control tissues (liver and pancreas) is shown.

FIGS. 12A-12E. SPARTA Methodology. (FIG. 12A) A naive human scFv library(˜10¹¹ TU) is first screened in vitro against immobilized recombinantantigens. The phage output pool (˜10⁶ TU) is subsequently transferred toa yeast display vector for two additional screening steps. After roundsof positive and negative sorting, the yeast output pool (˜10⁴ TU) isexpressed multivalently in phage particles and administered i.v. intotumor-bearing mice. Tumor-homing phage particles are recovered,amplified by PCR and re-expressed in multivalent format for twoadditional rounds of in vivo selection. Clonal diversity and ranking aredetermined after NGS. (FIGS. 12B and 12C) Flow cytometry profiles andELISA. Positive binders are shown for selected anti-EphA5 or anti-GRP78.Each dot in the FACS plot (top-right quadrant) represents an individualyeast antibody-displaying clone. ELISA carried out with thecorresponding recombinant antigen confirmed binding specificity. Opencircles represent individual data points. (FIGS. 12D and 12E) Followingthe screening steps in vitro, three rounds of selection in vivo wereperformed in mice bearing human lung cancer xenografts for EphA5, orisogenic mammary tumors for GRP78. Open circles represent individualdata points.

FIG. 13. Anti-Epha5 Monoclonal Antibodies Home To Human Lung CancerXenografts In Vivo. Selected anti-EphA5 antibody clones (termed E1-4)were tested individually for their ability to target EphA5-expressingtumors in a xenograft model of lung cancer (n=2). A representative graphis shown for each targeted phage clone. Phage displaying an antibodyfragment against the viral protein M2 was used as negative control.Experiments were performed at least twice with similar results. Arepresentative experiment is shown. Open circles represent individualdata points.

FIG. 14. Anti-GRP78 Monoclonal Antibodies Home To Isogenic Breast TumorsIn Vivo. Selected anti-GRP78 antibody clones (termed G1-3) were testedindividually for their ability to target an isogenic model of mammarycancer expressing cell surface GRP78 (n=2). A representative graph isshown for each targeted phage clone. Phage displaying an antibodyfragment against the viral protein M2 was used as a negative control.Experiments were performed at least twice with similar results. Arepresentative experiment is shown. Open circles represent individualdata points.

FIGS. 15A-15B. Characterization Of Scfv-Displaying Phage In Vitro (FIG.15A) Selected anti-EphA5 clones or anti-GRP78 clones were testedindividually for binding to recombinant antigens. Negative controlsincluded an anti-M2 clone and BSA. Open circles represent individualdata points. (FIG. 15B) Binding to antigens expressed on the cellsurface was tested by standard ELISA on cells. (FIG. 15B) EphA5-positive(H460) and EphA5-negative (H226) cells grown in 96-well plates wereexposed to anti-EphA5 clones or to a control phage. All four monoclonalantibodies bound specifically to H460 lung cancer cells. (FIG. 15B,lower panel) Similarly, all anti-GRP78 clones bound to GRP78-expressingbreast cancer cells (MCF7) whereas a control phage showed onlybackground binding.

FIG. 16. Negative Selection of a Polyclonal Pool of Anti-Epha5Antibodies against the Family Member Proteins EphA3, EphA4, EphA6 AndEphA7 Clones that showed no binding to the pooled EphA family proteinswere enriched by flow cytometry sorting. The resulting populationrevealed an antibody pool highly specific to EphA5, and not other familymembers.

FIG. 17. Validation of Anti-GRP78 scfv-Fc Binding Specificity In Vitro.Anti-GRP78 scFv-Fc agents produced in yeast were tested by ELISA (FIG.17, upper panel) and by flow cytometry (FIG. 17, lower panel) onEf43.fgf4 cells. All clones showed binding specificity when compared toan isotype negative control antibody. Open circles represent individualdata points.

FIGS. 18A-18B. Validation of Anti-Epha5 scfv-Fcs In Vitro. Theanti-EphA5 scFv-Fcs produced in CHO cells were tested by ELISA (A) andwhole-cell ELISA (B) on EphA5 positive (H460) or negative (H226) lungcancer cells. All clones show binding specificity when compared to anisotype negative control. Open circles represent individual data points.

FIG. 19. Tumor Targeting In Vivo. The anti-EphA5 clone E4 scFv-Fcproduced in CHO or the anti-GRP78 clone G1 scFv-Fc produced in S.cerevisia were administered i.v. into mice bearing human lung cancerxenografts or isogenic mammary tumor (n=2). After a circulation time nolonger than 6 min, tumors and control organs were collected andprocessed for immunofluorescence with a Cy3-conjugated anti-Fc. Tumortargeting was observed whereas only background staining was detectablein negative control organs. An unrelated scFv-Fc served as negativecontrol.

FIG. 20. Selection and Characterization of Tumor Cell Lines for In VitroCytotoxicity Assays The number of surface molecules was evaluated byflow cytometry-based quantitative analysis of antigen expression on thetumor cell surface. (FIG. 20, upper panel) The number of EphA5 moleculeson the tumor cell surface was quantified on a representative panel ofhuman lung cancer cell lines (n=4), as indicated. (FIG. 20, lower panel)Quantification of cell surface GRP78 was performed on Ef43.fgf4 breastcancer cells. GRP78-silenced Ef43.fgf4 cells served as control. Opencircles represent individual data points.

FIGS. 21A-21D. Complete Panel of Cytotoxic Profiles of Anti-Epha5 andAnti-GRP78 Monoclonal Antibodies Cytotoxicity was measured in real-timein presence of secondary Fab antibodies conjugated with AAMT, DMDM,MMAF, and DM1.

FIG. 22. Sequences of selected anti-EphA5 and anti-GRP78 HCDR3.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure provides targeting antibodies, antigen bindingfragments thereof, immunoconjugates comprising the foregoing, andtherapeutic methods for the treatment of cancers. In some aspects, theantibodies are human monoclonal anti-EphA5 antibodies or scFvs thereof,such as for the treatment of cancers expressing EphA5. The disclosurealso provides an effective strategy to generate highly specificantibodies which recognize the target antigen in its native conformationwithin the relevant physiological context.

I. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and CurrentProtocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons,Inc. 2001. As appropriate, procedures involving the use of commerciallyavailable kits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies, polyclonalantibodies, multivalent antibodies, and multispecific antibodies,regardless of how they are produced (i.e., using immunization,recombinant, synthetic methodologies).

The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as themyriad immunoglobulin variable region genes. Light chains are classifiedas either kappa or lambda. Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, which in turn define the immunoglobulinclasses, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about sss25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light” chain,domain, region and component are used interchangeably, are abbreviatedby “VL” or “V_(L)” and refer to the light chain of an antibody orantibody fragment. Similarly, terms “variable heavy” chain, domain,region and component are used interchangeably, are abbreviated by “VH”or “V_(H) ” and refer to the heavy chain of an antibody or antibodyfragment.

The terms “anti-EphA5 antibody”, “EphA5 antibody” and “EphA5-specificspecific antibody” are used interchangeably and refer to antibodies thatare specific for and bind specifically to EphA5. EphA5 antibodies of theinvention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

The terms “anti-GRP78 antibody”, “GRP78 antibody” and “GRP78-specificantibody” are used interchangeably and refer to antibodies that arespecific for and bind specifically to GRP78. GRP78 antibodies of theinvention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

As used herein, the terms “specific”, “specifically reactive”, “specificbinding”, “specifically binds” and “binds specifically” when used inconnection with EphA5 or GRP78 antibodies and antigen binding fragmentsthereof refer to the selective binding to EphA5 or GRP78, respectivelyas determined using standard immunological detection assays, includingwithout limitation ELISA, immunoblot, Western Blot, immunohistochemicaland immunoprecipitation assays, under conditions typically employed forconducting such assays. EphA5 or GRP78 antibodies and antigen bindingfragments thereof may be tested for such specificity using methods wellknown in the art and as described herein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988), for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably, to refer to an antibody in itssubstantially intact form, not as antibody fragments as defined below.The terms particularly refer to an antibody with heavy chains thatcontain the Fc region. A full length antibody can be a native sequenceantibody or an antibody variant.

“Antigen binding fragments” of antibodies comprise only a portion of anintact antibody, generally including an antigen binding site of theintact antibody and thus retaining the ability to bind antigen. Examplesof antibody fragments encompassed by the present definition include: (i)the Fab fragment, having VL, CL, VH and CH1 domains; (ii) the Fab′fragment, which is a Fab fragment having one or more cysteine residuesat the C-terminus of the CH1 domain; (iii) the Fd fragment having VH andCH1 domains; (iv) the Fd′ fragment having VH and CH1 domains and one ormore cysteine residues at the C-terminus of the CH1 domain; (v) the Fvfragment having the VL and VH domains of a single arm of an antibody;(vi) the dAb fragment which consists of a VH domain; (vii) isolated CDRregions; (viii) F(ab′)₂ fragments, a bivalent fragment including twoFab′ fragments linked by a disulfide bridge at the hinge region; (ix)single chain antibody molecules (e.g. single chain Fv; scFv); (x)“diabodies” with two antigen binding sites, comprising a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain; (xi) “linear antibodies” comprising a pairof tandem Fd, segments (VH-CH1-VH-CH1) which, together withcomplementary light chain polypeptides, form a pair of antigen bindingregions.

As used herein, the term “single-chain Fv” or “scFv” or “single chain”antibody refers to antibody fragments comprising th VH and VL domains ofantibody, wherein these domains are present in a single polypeptidechain. Generally, the Fv polypeptide further comprises a polypeptidelinker between the VH and VL domains which enables the sFv to form thedesired structure for antigen binding. For a review of sFv, seePluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. The monoclonal antibodies of the invention maybe generated by recombinant DNA methods, and are sometimes referred toas “recombinant antibodies” or “recombinant monoclonal antibodies”herein.

Recombinant antibody fragments may be isolated from phage antibodylibraries using techniques well known in the art. See, for example,Clackson et al., 1991, Nature 352: 624-628; Marks et al., 1991, J. Mol.Biol. 222: 581-597. Recombinant antibody fragments may be derived fromlarge phage antibody libraries generated by recombination in bacteria(Sblattero and Bradbury, 2000, Nature Biotechnology 18:75-80; and asdescribed herein). Polynucleotides encoding the VH and VL components ofantibody fragments (i.e., scFv) may be used to generate recombinant fulllength immunoglobulins using methods known in the art (see, for example,Persic et al., 1997, Gene 187: 9-18).

As used herein, “linker” or “spacer” refers to a molecule or group ofmolecules that connects two molecules, such as VH and VL genes orpolypeptides (i.e., in a scFv), and serves to place the two molecules ina preferred configuration.

The terms “label” and “detectable label” refer to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” or “detectably labeled” antibody.The label may be detectable by itself (e.g. radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable. A great number of such labels are known in the art,including without limitation protein tags, radioisotopes, metalchelators, enzymes, fluorescent compounds (dyes, proteins, chemicals),bioluminescent compounds, and chemiluminescent compounds.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, a nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a nucleic acid encoding afluorescent protein from one source and a nucleic acid encoding apeptide sequence from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “display vector” refers to a vector used to create a cell or virusthat displays, i.e., expresses a display protein comprising aheterologous polypeptide, on its surface or in a cell compartment suchthat the polypeptide is accessible to test binding to target moleculesof interest, such as antigens.

A “display library” refers to a population of display vehicles, often,but not always, cells or viruses. The “display vehicle” provides boththe nucleic acid encoding a peptide as well as the peptide, such thatthe peptide is available for binding to a target molecule and further,provides a link between the peptide and the nucleic acid sequence thatencodes the peptide. Various “display libraries” are known to those ofskill in the art and include libraries such as phage, phagemids, yeastand other eukaryotic cells, bacterial display libraries, plasmid displaylibraries as well as in vitro libraries that do not require cells, forexample ribosome display libraries or mRNA display libraries, where aphysical linkage occurs between the mRNA or cDNA nucleic acid, and theprotein encoded by the mRNA or cDNA.

A “phage expression vector” or “phagemid” refers to any phage-basedrecombinant expression system for the purpose of expressing a nucleicacid sequence in vitro or in vivo, constitutively or inducibly, in anycell, including prokaryotic, yeast, fungal, plant, insect or mammaliancell. A phage expression vector typically can both reproduce in abacterial cell and, under proper conditions, produce phage particles.The term includes linear or circular expression systems and encompassesboth phage-based expression vectors that remain episomal or integrateinto the host cell genome.

A “phage display library” refers to a “library” of bacteriophages onwhose surface is expressed exogenous peptides or proteins. The foreignpeptides or polypeptides are displayed on the phage capsid outersurface. The foreign peptide can be displayed as recombinant fusionproteins incorporated as part of a phage coat protein, as recombinantfusion proteins that are not normally phage coat proteins, but which areable to become incorporated into the capsid outer surface, or asproteins or peptides that become linked, covalently or not, to suchproteins. This is accomplished by inserting an exogenous nucleic acidsequence into a nucleic acid that can be packaged into phage particles.Such exogenous nucleic acid sequences may be inserted, for example, intothe coding sequence of a phage coat protein gene. If the foreignsequence is “in phase” the protein it encodes will be expressed as partof the coat protein. Thus, libraries of nucleic acid sequences, such asa genomic library from a specific cell or chromosome, can be so insertedinto phages to create “phage libraries.” As peptides and proteinsrepresentative of those encoded for by the nucleic acid library aredisplayed by the phage, a “peptide-display library” is generated. Whilea variety of bacteriophages are used in such library constructions,typically, filamentous phage are used (Dunn, 1996 Curr. Opin.Biotechnol. 7:547-553). See, e.g., description of phage displaylibraries, below.

A “yeast display library” refers to a “library of yeast on whose surfaceis expressed exogenous peptides or proteins. A yeast surface display isan alternative method for isolating and engineering antibody fragments(scFv, Fab, Fab′, F(ab′)2, Fd, Fv, dAb, preferably isolated scFv) fromimmune and non-immune libraries, and has been used to isolaterecombinant antibodies with binding specificity to a variety of proteinsand peptides. In the yeast display system, yeast display libraries areproduced by cloning the pool of genes coding for antibody fragments intovectors that can be transformed into the yeast. The transformed antibodygenes encoding for exogenous peptides or proteins are displayed on thesurface of the yeast, often via fusion to an a-agglutinin yeast adhesionreceptor, which is located in the yeast cell wall. Like phage display,yeast display provides a direct connection between genotype andphenotype. A plasmid containing the gene of interest is contained withinyeast cells and the encoded antibody is expressed on the surface. Thedisplay level of each yeast cell is variable, with each cell displayingfrom approximately 1×10⁴ to 1×10⁵ copies of the scFv. There are usually3-5 rounds of enrichment of target antigen-binding clones. The antibodyexpressed by a single yeast colony is evaluated for specificity andreproducibility. If the antibody has the required functionality, theantibody gene is sequenced as part of antibody validation. Screening ofantibody variation of surface expression and avidity can be quantifiedusing fluorescence activated cell sorting (FACS), which measures boththe strength of antigen binding and amount of antibody expression on theyeast cell surface using separate tags on the antibody and antigen. FACSbinding assays provide a much more quantitative way of assessing highbinding affinity and selectivity for the antigens and offer the abilityto accurately control selection parameters (binding populationpercentage, signal normalization, and desired binding affinities) byflow cytometry.

“Serial screening” or “screened serially” is a term used to describe thescreening of a phage, yeast, phagemid or other library against anantigen, preferably an immobilized antigen pursuant to the SPART methoddescribed herein where the library is screened, bound particles of thelibrary are then isolated and collected and the isolated particles arescreened against the same antigen (which may be the same or preferably adifferent sample of the same antigen). This serial screening processwill occur at least twice and in many in instances as many as 3-10,often 3-6 times to provide a final pool of particles, the geneticmaterial of which can be isolated, cloned and introduced into analternative library for further screening. In most instances the serialscreening process occurs without isolating the DNA from the particleswhich have been screened. In other instances, the DNA from the particleswhich have been screened are isolated and recloned into a naive displayvector used in the first screen before each subsequent screening.

The term “cloning” is used to describe the methods of isolating,amplifying and transferring/inicorporating genetic material expressingexogenous proteins or peptides identified through screening from onedisplay vector (e.g. a phage display vector) into an unmodified displayvector (which may be same type of display vector or a different type ofdisplay vector) for further screening or an expression vector forexpressing the identified protein or peptide (e.g., to produce antibodyfragments identified through the screening methodology). Cloningtechniques which are used in the present invention are well known in theart. See, for example the following references, which are incorporatedby reference herein. Andris-Widhopf, et al., (2011); Barbas, et a;.(2001); Ferrara, et al., (2012); Hoogenboom, et a.1, (1991); Huston,etal., (1988); Krebber, etal., (1997); Marks, J. D., & Bradbury, A.(2004); and Schaefer, et al., (2010).

IIA. EphA5 Antibodies

Eph receptor tyrosine kinases and their ligands (ephrin) regulate a widerange of cell contact-dependent signaling that can effect cellproliferation, migration, morphology, adhesion, and invasion (Pitulescuet al. Genes & Dev., 24:2480-2492, 2010). However, elevated EphA5 hasalso been found in variety of cancer cell types, such as lung cancer andbrain cancer. Studies herein demonstrate that inhibition of EphA5activity is effective for inhibiting cancer cell proliferation andangiogenesis in tumor tissues. Moreover, EphA5-binding antibodiesprovided here were found to be effective for inhibiting EphA5 activityand cancer cell growth. Thus, antibodies of the embodiments provide neweffective methods for treating cancers and inhibiting angiogenesis.

In some embodiments, the present disclosure provides anti-EphA5 humanmonoclonal antibodies. These antsibodies are selected from extremelylarge recombinant naive human antibody libraries by using a combinationof phage and yeast display. This combined approach allows for theselection of populations of high affinity binders as well as theelimination of antibodies with undesired cross-reactivities. Inaddition, the in vitro selected population is further selected by meansof a in vivo selection strategy in mice bearing xenograft tumors usingboth mono- and multivalent phage display.

The phage antibody clones recovered from the tumor tissue have twodesirable properties. First, they have tumor homing capacity. Second,they recognize the target in its in vivo conformation and physiologicalcontext. Tumor homing antibody clones generated using the methods of theinvention are identified through their HCDR3 using next generationsequencing and a tailored bioinformatics analysis. Antibody clones areranked by abundance and the top ranking clones are then rescued by aninverse-PCR based strategy and produced as single clones. Selectedclones may then be evaluated for cell binding and cell internalizationproperties, and ultimately for cytotoxicity.

Among the various aspects of the invention, anti-EphA5 antibodies areprovided. The anti-EphA5 antibodies of the invention are specific forEphA5. Preferred anti-EphA5 antibodies are isolated, purified orsemi-purified such that they retain specificity in the desiredapplication. Most preferred for therapeutic applications in thetreatment of cancers expressing EphA5 are fully human monoclonalantibodies.

Another aspect of the invention relates to antigen binding fragments ofEphA5 antibodies which are specific for EphA5. Such fragments may begenerated from intact antibodies or through the use of recombinanttechnology. For example, an EphA5 antibody antigen binding fragment maybe a single chain antibody, or scFv. In one embodiment, an EphA5 humanmonoclonal antibody or antigen binding fragment thereof comprises theHCDR3 amino acid sequence of SEQ ID NOs: 5, 13, 21, or 29. In anotherembodiment, an EphA5 human monoclonal antibody or antigen bindingfragment thereof comprises a heavy chain variable region having an aminoacid sequence that is at least 80%, preferably about 90%, 91%, 92%, 93%or 94%, and most preferably about 95% or more, identical to SEQ ID NOs:1, 9, 17, or 25. In another embodiment, an EphA5 human monoclonalantibody of the invention comprises a light chain variable region havingan amino acid sequence that is at least 80%, preferably about 90%, 91%,92%, 93% or 94%, and most preferably about 95% or more, identical to SEQID NOs: 2, 10, 18, or 26. The EphA5 antibodies of the invention may beof the immunoglobulin classes IgA, IgD, IgE, IgG and IgM and subclassesthereof.

In one embodiment, phage display systems are used to select single chainantibodies specific for EphA5. Once isolated, polynucleotides encodingthe EphA5 scFvs may be cloned into expression vectors designed toexpress full length immunoglobulins as well as fragments thereof havingthe same specificity. Briefly, to generate a full length antibody, theVH and VL genes of the single chain antibody are cloned into animmunoglobulin scaffold (i.e., IgG) vector, expressed, and dimerized inorder to convert the single chain into a full antibody. Theimmunoglobulin scaffold may be selected from any of the five majorclasses of immunoglobulins (IgA, IgD, IgE, IgG and IgM), and subclassesthereof (i.e., IgG-1). Exemplary selection and screening strategies aredescribed in the Examples,

EphA5 antibodies and antigen binding fragment thereof may be detectablylabeled as is generally known. The label may be detectable by itself(e.g. radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable. A great number of suchlabels are known in the art, including without limitation protein tags,radioisotopes, metal chelators, enzymes, fluorescent compounds (dyes,proteins, chemicals), bioluminescent compounds, and chemiluminescentcompounds. Such labeled EphA5 antibodies and antigen binding fragmentsthereof may be used, for example, to immunologically detect or imageEphA5 expressing cells and tumors.

IIB. GRP78 Antibodies

In some embodiments, the present disclosure concerns anti-GRP78 humanmonoclonal antibodies. These antibodies are selected from extremelylarge recombinant naive human antibody libraries by using a combinationof phage and yeast display. This combined approach allows for theselection of populations of high affinity binders as well as theelimination of antibodies with undesired cross-reactivities. Inaddition, the in vitro selected population is further selected by meansof an in vivo selection strategy in mice bearing xenograft tumors usingboth mono- and multivalent phage display.

The phage antibody clones recovered from the tumor tissue have twodesirable properties. First, they have tumor homing capacity. Second,they recognize the target in its in vivo conformation and physiologicalcontext. Tumor homing antibody clones generated using the methods of theinvention are identified through their HCDR3 using next generationsequencing and a tailored bioinformatics analysis. Antibody clones areranked by abundance and the top ranking clones are then rescued by aninverse-PCR based strategy and produced as single clones. Selectedclones may then be evaluated for cell binding and cell internalizationproperties, and ultimately for cytotoxicity.

Among the various aspects of the invention, anti-GRP78 antibodies areprovided. The anti-GRP78 antibodies of the invention are specific forGRP78. Preferred anti-GRP78 antibodies are isolated, purified orsemi-purified such that they retain specificity in the desiredapplication. Most preferred for therapeutic applications in thetreatment of cancers expressing GRP78 are fully human monoclonalantibodies.

Another aspect of the invention relates to antigen binding fragments ofGRP78 antibodies which are specific for GRP78. Such fragments may begenerated from intact antibodies or through the use of recombinanttechnology. For example, an GRP78 antibody antigen binding fragment maybe a single chain antibody, or scFv. In one embodiment, an GRP78 humanmonoclonal antibody or antigen binding fragment thereof comprises theHCDR3 of SEQ ID NO: 41, SEQ ID NO: 49, or SEQ ID NO: 57. In anotherembodiment, an GRP78 human monoclonal antibody or antigen bindingfragment thereof comprises a heavy chain variable region having an aminoacid sequence that is at least 80%, preferably about 90%, 91%, 92%, 93%or 94%, and most preferably about 95% or more, identical to SEQ ID NO:37, SEQ ID NO: 45, or SEQ ill) NO: 53. In another embodiment, an GRP78human monoclonal antibody of the invention comprises a light chainvariable region having an amino acid sequence that is at least 80%,preferably about 90%, 91%, 92%, 93% or 94%, and most preferably about95% or more, identical to SEQ ID NO: 38, SEQ ID NO: 46, or SEQ LD NO:54. The GRP78 antibodies of the invention may be of the immunoglobulinclasses IgA, IgD, IgE, IgG and IgM and sub classes thereof.

In one embodiment, phage display systems are used to select single chainantibodies specific for GRP78. Once isolated, polynucleotides encodingthe GRP78 scFvs may be cloned into expression vectors designed toexpress full length immunoglobulins as well as fragments thereof havingthe same specificity. Briefly, to generate a full length antibody, theVH and VL genes of the single chain antibody are cloned into animmunoglobulin scaffold (i.e., IgG) vector, expressed, and dimerized inorder to convert the single chain into a full antibody. Theimmunoglobulin scaffold may be selected from any of the five majorclasses of immunoglobulins (IgA, IgD, IgE, IgG and IgM), and subclassesthereof (i.e., IgG-1). Exemplary selection and screening strategies aredescribed in the Examples, infra.

GRP78 antibodies and antigen binding fragment thereof may be detectablylabeled as is generally known. The label may be detectable by itself(e.g. radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable. A great number of suchlabels are known in the art, including without limitation protein tags,radioisotopes, metal chelators, enzymes, fluorescent compounds (dyes,proteins, chemicals), bioluminescent compounds, and chemiluminescentcompounds. Such labeled GRP78 antibodies and antigen binding fragmentsthereof may be used, for example, to immunologically detect or imageGRP78 expressing cells and tumors.

Another aspect of the invention relates to polyriucloetides encoding theEphA5 or GRP78 antibodies and antigen binding fragments of theinvention, and includes vectors comprising such polynucleotides as wellas host cells comprising such vectors.

In yet another aspect, the invention provides therapeutic compositions,including without limitation immunoconjugates, such as antibody-drugconjugates (ADC), which comprise an anti-EphA5 or anti-GRP78 antibody orantigen binding fragment thereof conjugated to a cytotoxic agent such asa chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin, ora radioactive isotope. In related aspect, the invention further providesmethods of using the immunoconjugates for the treatment of patients withneoplasms expressing EphA5 or GRP78 respectively. Antibody-drugconjugates are a new class of highly potent biopharmaceutical drugsdesigned as a targeted therapy for the treatment of patients withcancer. ADCs are complex molecules comprising an antibody or an antibodyfragment linked via a stable linker to a biological active cytotoxicpayload or drug. By combining the unique targeting capabilities ofmonoclonal antibodies with the cancer-killing ability of cytotoxicdrugs, antibody-drug conjugates allow sensitive discrimination betweenhealthy and diseased tissue.

In certain embodiments, an antibody or a fragment thereof that binds toat least a portion of EphA5 or GPR78 protein and inhibits EphA5 or GPR78signaling and cancer cell proliferation are contemplated. The antibodymay be any immunologic binding agent, such as IgG, IgM, IgA, IgD, IgE,and genetically modified IgG as well as polypeptides comprising antibodyCDR domains that retain antigen binding activity. The antibody may beselected from the group consisting of a chimeric antibody, an affinitymatured antibody, a polyclonal antibody, a monoclonal antibody, ahumanized antibody, a human antibody, or an antigen-binding antibodyfragment or a natural or synthetic ligand. Preferably, the anti-EphA5 oranti-GRP78 antibody is a monoclonal antibody or a humanized antibody.

Thus, by known means and as described herein, polyclonal or monoclonalantibodies, antibody fragments, and binding domains and CDRs (includingengineered forms of any of the foregoing) may be created that arespecific to EphA5 or GRP78 protein, one or more of its respectiveepitopes, or conjugates of any of the foregoing, whether such antigensor epitopes are isolated from natural sources or are syntheticderivatives or variants of the natural compounds.

Examples of antibody fragments suitable for the present embodimentsinclude, without limitation: (i) the Fab fragment, consisting of V_(L),V_(H), C_(L), and C_(H1) domains; (ii) the “Fd” fragment consisting ofthe V_(H) and Cm domains; (iii) the “Fv” fragment consisting of theV_(L) and V_(H) domains of a single antibody; (iv) the “dAb” fragment,which consists of a V_(H) domain; (v) isolated CDR regions; (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments;(vii) single chain Fv molecules (“scFv”), wherein a V_(H) domain and aV_(L) domain are linked by a peptide linker that allows the two domainsto associate to form a binding domain; (viii) bi-specific single chainFv dimers (see U.S. Pat. No. 5,091,513); and (ix) diabodies, multivalentor multispecific fragments constructed by gene fusion (US Patent App.Pub. 20050214860). Fv, scFv, or diabody molecules may be stabilized bythe incorporation of disulphide bridges linking the V_(H) amd V_(L)domains. Minibodies comprising a scFv joined to a CH3 domain may also bemade (Hu et al., 1996).

Antibody-like binding peptidomimetics are also contemplated inembodiments. Liu et al. (2003) describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods.

Animals may be inoculated with an antigen, such as a EphA5 or GRP78extracellular domain (ECD) protein, in order to produce antibodiesspecific for EphA5 or GRP 78 protein. Frequently an antigen is bound orconjugated to another molecule to enhance the immune response. As usedherein, a conjugate is any peptide, polypeptide, protein, ornon-proteinaceous substance bound to an antigen that is used to elicitan immune response in an animal. Antibodies produced in an animal inresponse to antigen inoculation comprise a variety of non-identicalmolecules (polyclonal antibodies) made from a variety of individualantibody producing B lymphocytes. A polyclonal antibody is a mixedpopulation of antibody species, each of which may recognize a differentepitope on the same antigen. Given the correct conditions for polyclonalantibody production in an animal, most of the antibodies in the animal'sserum will recognize the collective epitopes on the antigenic compoundto which the animal has been immunized. This specificity is furtherenhanced by affinity purification to select only those antibodies thatrecognize the antigen or epitope of interest.

A monoclonal antibody is a single species of antibody wherein everyantibody molecule recognizes the same epitope because all antibodyproducing cells are derived from a single B-lymphocyte cell line. Themethods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies. Insome embodiments, rodents such as mice and rats are used in generatingmonoclonal antibodies. In some embodiments, rabbit, sheep, or frog cellsare used in generating monoclonal antibodies. The use of rats is wellknown and may provide certain advantages. Mice (e.g., BALB/c mice) areroutinely used and generally give a high percentage of stable fusions.

Hybridoma technology involves the fusion of a single B lymphocyte from amouse previously immunized with a EphA5 or GRP78 antigen with animmortal myeloma cell (usually mouse myeloma). This technology providesa method to propagate a single antibody-producing cell for an indefinitenumber of generations, such that unlimited quantities of structurallyidentical antibodies having the same antigen or epitope specificity(monoclonal antibodies) may be produced.

Plasma B cells (CD45+CD5−CD19+) may be isolated from freshly preparedmouse peripheral blood mononuclear cells of immunized mice and furtherselected for EphA5 or GRP78 binding cells. After enrichment of antibodyproducing B cells, total RNA may be isolated and cDNA synthesized. DNAsequences of antibody variable regions from both heavy chains and lightchains may be amplified, constructed into a phage display Fab expressionvector, and transformed into E. coli. EphA5 or GRP78 specific bindingFab may be selected out through multiple rounds enrichment panning andsequenced. Selected EphA5 or GRP78 binding hits may be expressed as fulllength IgG in mouse and mouse/human chimeric forms using a mammalianexpression vector system in human embryonic kidney (HEK293) cells(Invitrogen) and purified using a protein G resin with a fast proteinliquid chromatography (FPLC) separation unit.

In one embodiment, the antibody is a chimeric antibody, for example, anantibody comprising antigen binding sequences from a non-human donorgrafted to a heterologous non-human, human, or humanized sequence (e.g.,framework and/or constant domain sequences). Methods have been developedto replace light and heavy chain constant domains of the monoclonalantibody with analogous domains of human origin, leaving the variableregions of the foreign antibody intact. Alternatively, “fully human”monoclonal antibodies are produced in mice transgenic for humanimmunoglobulin genes. Methods have also been developed to convertvariable domains of monoclonal antibodies to more human form byrecombinantly constructing antibody variable domains having both rodent,for example, mouse, and human amino acid sequences. In “humanized”monoclonal antibodies, only the hypervariable CDR is derived from mousemonoclonal antibodies, and the framework and constant regions arederived from human amino acid sequences (see U.S. Pat. Nos. 5,091,513and 6,881,557). It is thought that replacing amino acid sequences in theantibody that are characteristic of rodents with amino acid sequencesfound in the corresponding position of human antibodies will reduce thelikelihood of adverse immune reaction during therapeutic use. Ahybridoma or other cell producing an antibody may also be subject togenetic mutation or other changes, which may or may not alter thebinding specificity of antibodies produced by the hybridoma.

Methods for producing polyclonal antibodies in various animal species,as well as for producing monoclonal antibodies of various types,including humanized, chimeric, and fully human, are well known in theart and highly predictable. For example, the following U.S. patents andpatent applications provide enabling descriptions of such methods: U.S.Patent Application Nos. 2004/0126828 and 2002/0172677; and U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149;4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003;4,742,159; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778;5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,253;5,565,332; 5,571,698; 5,627,052; 5,656,434; 5,770,376; 5,789,208;5,821,337; 5,844,091; 5,858,657; 5,861,155; 5,871,907; 5,969,108;6,054,297; 6,165,464; 6,365,157; 6,406,867; 6,709,659; 6,709,873;6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; and 6,891,024.All patents, patent application publications, and other publicationscited herein and therein are hereby incorporated by reference in thepresent application.

Antibodies may be produced from any animal source, including birds andmammals. Preferably, the antibodies are ovine, murine (e.g., mouse andrat), rabbit, goat, guinea pig, camel, horse, or chicken. In addition,newer technology permits the development of and screening for humanantibodies from human combinatorial antibody libraries. For example,bacteriophage antibody expression technology allows specific antibodiesto be produced in the absence of animal immunization, as described inU.S. Pat. No. 6,946,546, which is incorporated herein by reference.These techniques are further described in: Marks (1992); Stemmer (1994);Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).

It is fully expected that antibodies to EphA5 or GRP78 will have theability to neutralize or counteract the effects of EphA5 or GRP78respectively regardless of the animal species, monoclonal cell line, orother source of the antibody. Certain animal species may be lesspreferable for generating therapeutic antibodies because they may bemore likely to cause allergic response due to activation of thecomplement system through the “Fc” portion of the antibody. However,whole antibodies may be enzymatically digested into “Fc” (complementbinding) fragment, and into antibody fragments having the binding domainor CDR. Removal of the Fc portion reduces the likelihood that theantigen antibody fragment will elicit an undesirable immunologicalresponse, and thus, antibodies without Fc may be preferential forprophylactic or therapeutic treatments. As described above, antibodiesmay also be constructed so as to be chimeric or partially or fullyhuman, so as to reduce or eliminate the adverse immunologicalconsequences resulting from administering to an animal an antibody thathas been produced in, or has sequences from, other species.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

Proteins may be recombinant, or synthesized in vitro, although in mostembodiments, the proteins are recombinant. Alternatively, anon-recombinant or recombinant protein may be isolated from bacteria. Itis also contemplated that a bacteria containing such a variant may beimplemented in compositions and methods. Consequently, a protein neednot be isolated.

It is contemplated that in compositions there is between about 0.001 mgand about 10 mg of total polypeptide, peptide, and/or protein per ml.Thus, the concentration of protein in a composition can be about, atleast about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or anyrange derivable therein). Of this, about, at least about, or at mostabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% may be an antibody that bindsEphA5 or GRP78.

An antibody or preferably an immunological portion of an antibody, canbe chemically conjugated to, or expressed as, a fusion protein withother proteins. For purposes of this specification and the accompanyingclaims, all such fused proteins are included in the definition ofantibodies or an immunological portion of an antibody.

Embodiments provide antibodies and antibody-like molecules against EphA5or GRP78, polypeptides and peptides that are linked to at least oneagent to form an antibody conjugate or payload. In order to increase theefficacy of antibody molecules as diagnostic or therapeutic agents, itis conventional to link or covalently bind or complex at least onedesired molecule or moiety. Such a molecule or moiety may be, but is notlimited to, at least one effector or reporter molecule. Effectormolecules comprise molecules having a desired activity, e.g., cytotoxicactivity. Non-limiting examples of effector molecules that have beenattached to antibodies include toxins, therapeutic enzymes, antibiotics,radio-labeled nucleotides and the like. By contrast, a reporter moleculeis defined as any moiety that may be detected using an assay.Non-limiting examples of reporter molecules that have been conjugated toantibodies include enzymes, radiolabel s, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,luminescent molecules, photoaffinity molecules, colored particles orligands, such as biotin.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3-6-diphenylglycouril-3 attached to the antibody.Monoclonal antibodies may also be reacted with an enzyme in the presenceof a coupling agent such as glutaraldehyde or periodate. Conjugates withfluorescein markers are prepared in the presence of these couplingagents or by reaction with an isothiocyanate.

III. Treatment of Diseases

Certain aspects of the present embodiments can be used to prevent ortreat a disease or disorder associated with EphA5 or GRP78 signaling.Signaling of EphA5 or GRP78 may be reduced by any suitable drugs toprevent cancer cell proliferation. Preferably, such substances would bean anti-EphA5 antibody.

“Treatment” and “treating” refer to administration or application of atherapeutic agent to a subject or performance of a procedure or modalityon a subject for the purpose of obtaining a therapeutic benefit of adisease or health-related condition. For example, a treatment mayinclude administration of a pharmaceutically effective amount of anantibody that inhibits the EphA5 or FRP78 signaling.

“Subject” and “patient” refer to either a human or non-human, such asprimates, mammals, and vertebrates. In particular embodiments, thesubject is a human.

The term “therapeutic benefit” or “therapeutically effective” as usedthroughout this application refers to anything that promotes or enhancesthe well-being of the subject with respect to the medical treatment ofthis condition. This includes, but is not limited to, a reduction in thefrequency or severity of the signs or symptoms of a disease. Forexample, treatment of cancer may involve, for example, a reduction inthe size of a tumor, a reduction in the invasiveness of a tumor,reduction in the growth rate of the cancer, or prevention of metastasis.Treatment of cancer may also refer to prolonging survival of a subjectwith cancer.

A. Pharmaceutical Preparations

Where clinical application of a therapeutic composition containing aninhibitory antibody is undertaken, it will generally be beneficial toprepare a pharmaceutical or therapeutic composition appropriate for theintended application. In certain embodiments, pharmaceuticalcompositions may comprise, for example, at least about 0.1% of an activecompound. In other embodiments, an active compound may comprise betweenabout 2% to about 75% of the weight of the unit, or between about 25% toabout 60%, for example, and any range derivable therein.

The therapeutic compositions of the present embodiments areadvantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. These preparations also may be emulsified.

The phrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic, or other untoward reaction when administered to an animal,such as a human, as appropriate. The preparation of a pharmaceuticalcomposition comprising an antibody or additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure. Moreover, for animal (e.g., human) administration, it willbe understood that preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall aqueous solvents (e.g., water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles, such as sodium chloride, Ringer'sdextrose, etc.), non-aqueous solvents (e.g., propylene glycol,polyethylene glycol, vegetable oil, and injectable organic esters, suchas ethyloleate), dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial or antifungal agents, anti-oxidants,chelating agents, and inert gases), isotonic agents, absorption delayingagents, salts, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, fluid and nutrient replenishers, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart. The pH and exact concentration of the various components in apharmaceutical composition are adjusted according to well-knownparameters.

The term “unit dose” or “dosage” refers to physically discrete unitssuitable for use in a subject, each unit containing a predeterminedquantity of the therapeutic composition calculated to produce thedesired responses discussed above in association with itsadministration, i.e., the appropriate route and treatment regimen. Thequantity to be administered, both according to number of treatments andunit dose, depends on the effect desired. The actual dosage amount of acomposition of the present embodiments administered to a patient orsubject can be determined by physical and physiological factors, such asbody weight, the age, health, and sex of the subject, the type ofdisease being treated, the extent of disease penetration, previous orconcurrent therapeutic interventions, idiopathy of the patient, theroute of administration, and the potency, stability, and toxicity of theparticular therapeutic substance. For example, a dose may also comprisefrom about 1 ug/kg/body weight to about 1000 mg/kg/body weight (thissuch range includes intervening doses) or more per administration, andany range derivable therein. In non-limiting examples of a derivablerange from the numbers listed herein, a range of about 5 μg/kg/bodyweight to about 100 mg/kg/body weight, about 5 μg/kg/body weight toabout 500 mg/kg/body weight, etc., can be administered. The practitionerresponsible for administration will, in any event, determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject.

The active compounds can be formulated for parenteral administration,e.g., formulated for injection via the intravenous, intramuscular,sub-cutaneous, intravaginal or even intraperitoneal routes. Typically,such compositions can be prepared as either liquid solutions orsuspensions; solid forms suitable for use to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and, the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The proteinaceous compositions may be formulated into a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

A pharmaceutical composition can include a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion, and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin. clp B. CombinationTreatments

In certain embodiments, the compositions and methods of the presentembodiments involve an antibody or an antibody fragment against EphA5and/or GRP78 to inhibit its activity in cancer cell proliferation, incombination with a second or additional therapy. Such therapy can beapplied in the treatment of any disease that is associated with EphA5-or GRP78-mediated cell proliferation. For example, the disease may becancer.

The methods and compositions, including combination therapies, enhancethe therapeutic or protective effect, and/or increase the therapeuticeffect of another anti-cancer or anti-hyperproliferative therapy.Therapeutic and prophylactic methods and compositions can be provided ina combined amount effective to achieve the desired effect, such as thekilling of a cancer cell and/or the inhibition of cellularhyperproliferation. This process may involve contacting the cells withboth an antibody or antibody fragment and a second therapy. A tissue,tumor, or cell can be contacted with one or more compositions orpharmacological formulation(s) comprising one or more of the agents(i.e., antibody or antibody fragment or an anti-cancer agent), or bycontacting the tissue, tumor, and/or cell with two or more distinctcompositions or formulations, wherein one composition provides 1) anantibody or antibody fragment, 2) an anti-cancer agent, or 3) both anantibody or antibody fragment and an anti-cancer agent. Also, it iscontemplated that such a combination therapy can be used in conjunctionwith chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing, for example, both agents are delivered to a cellin a combined amount effective to kill the cell or prevent it fromdividing.

An inhibitory antibody may be administered before, during, after, or invarious combinations relative to an anti-cancer treatment. Theadministrations may be in intervals ranging from concurrently to minutesto days to weeks. In embodiments where the antibody or antibody fragmentis provided to a patient separately from an anti-cancer agent, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the two compounds wouldstill be able to exert an advantageously combined effect on the patient.In such instances, it is contemplated that one may provide a patientwith the antibody therapy and the anti-cancer therapy within about 12 to24 or 72 h of each other and, more particularly, within about 6-12 h ofeach other. In some situations it may be desirable to extend the timeperiod for treatment significantly where several days (2, 3, 4, 5, 6, or7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respectiveadministrations.

In certain embodiments, a course of treatment will last 1-90 days ormore (this such range includes intervening days). It is contemplatedthat one agent may be given on any day of day 1 to day 90 (this suchrange includes intervening days) or any combination thereof, and anotheragent is given on any day of day 1 to day 90 (this such range includesintervening days) or any combination thereof. Within a single day(24-hour period), the patient may be given one or multipleadministrations of the agent(s). Moreover, after a course of treatment,it is contemplated that there is a period of time at which noanti-cancer treatment is administered. This time period may last 1-7days, and/or 1-5 weeks, and/or 1-12 months or more (this such rangeincludes intervening days), depending on the condition of the patient,such as their prognosis, strength, health, etc. It is expected that thetreatment cycles would be repeated as necessary.

Various combinations may be employed. For the example below an antibodytherapy is “A” and an anti-cancer therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of any compound or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring toxicitythat is attributable to combination therapy.

i. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe present embodiments. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammall andcalicheamicin omegaI1); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and angui dine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine,plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids, or derivatives of any of theabove,

ii. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated, such as microwaves, proton beamirradiation (U.S. Pat. Nos 5,760,395 and 4,870,287), and UV-irradiation.It is most likely that all of these factors affect a broad range ofdamage on DNA, on the precursors of DNA, on the replication and repairof DNA, and on the assembly and maintenance of chromosomes. Dosageranges for X-rays range from daily doses of 50 to 200 roentgens forprolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000roentgens. Dosage ranges for radioisotopes vary widely, and depend onthe half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

iii. Immunotherapy

The skilled artisan will understand that additional immunotherapies maybe used in combination or in conjunction with methods of theembodiments. In the context of cancer treatment, immunotherapeutics,generally, rely on the use of immune effector cells and molecules totarget and destroy cancer cells. Rituximab (RITUXAN®) is such anexample. The immune effector may be, for example, an antibody specificfor some marker on the surface of a tumor cell. The antibody alone mayserve as an effector of therapy or it may recruit other cells toactually affect cell killing. The antibody also may be conjugated to adrug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and serve as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells

Antibody-drug conjugates have emerged as a breakthrough approach to thedevelopment of cancer therapeutics. Cancer is one of the leading causesof deaths in the world. Antibody—drug conjugates (ADCs) comprisemonoclonal antibodies (MAbs) that are covalently linked to cell-killingdrugs (FIG. 1). This approach combines the high specificity of MAbsagainst their antigen targets with highly potent cytotoxic drugs,resulting in “armed” MAbs that deliver the payload (drug) to tumor cellswith enriched levels of the antigen (Carter et al., 2008; Teicher 2014;Leal et al., 2014). Targeted delivery of the drug also minimizes itsexposure in normal tissues, resulting in decreased toxicity and improvedtherapeutic index. The approval of two ADC drugs, ADCETRIS® (brentuximabvedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013by FDA validated the approach. There are currently more than 30 ADC drugcandidates in various stages of clinical trials for cancer treatment(Leal et al., 2014). As antibody engineering and linker-payloadoptimization are becoming more and more mature, the discovery anddevelopment of new ADCs are increasingly dependent on the identificationand validation of new targets that are suitable to this approach(Teicher 2009) and the generation of targeting MAbs. Two criteria forADC targets are upregulated/high levels of expression in tumor cells androbust internalization.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present embodiments. Common tumormarkers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor,erb B, and p155. An alternative aspect of immunotherapy is to combineanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines, such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growthfactors, such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998);cytokine therapy, e.g., interferons α, β, and γ, IL-1, GM-CSF, and TNF(Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998);gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998;Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-gangliosideGM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat.No. 5,824,311). It is contemplated that one or more anti-cancertherapies may be employed with the antibody therapies described herein.

iv. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asthe treatment of the present embodiments, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy, and/or alternativetherapies. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, andmicroscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

v. Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present embodiments to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with certain aspectsof the present embodiments to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present embodiments. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent embodiments to improve the treatment efficacy.

IV. Kits and Diagnostics

In various aspects of the embodiments, a kit is envisioned containingtherapeutic agents and/or other therapeutic and delivery agents. In someembodiments, the present embodiments contemplates a kit for preparingand/or administering a therapy of the embodiments. The kit may compriseone or more sealed vials containing any of the pharmaceuticalcompositions of the present embodiments. The kit may include, forexample, at least one EphA5 antibody as well as reagents to prepare,formulate, and/or administer the components of the embodiments orperform one or more steps of the inventive methods. In some embodiments,the kit may also comprise a suitable container, which is a containerthat will not react with components of the kit, such as an eppendorftube, an assay plate, a syringe, a bottle, or a tube. The container maybe made from sterilizable materials such as plastic or glass.

The kit may further include an instruction sheet that outlines theprocedural steps of the methods set forth herein, and will followsubstantially the same procedures as described herein or are known tothose of ordinary skill in the art. The instruction information may bein a computer readable media containing machine-readable instructionsthat, when executed using a computer, cause the display of a real orvirtual procedure of delivering a pharmaceutically effective amount of atherapeutic agent.

V. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1— Generation of Anti-EphA5 Specific scFv Antibodies

In vitro scFv antibody selection: Recombinant human EphA5 antigen (R&DBiosystems) was biotinylated (EZ-Link™ Sulfo-NHS-LC-LC-Biotin, LifeTechnologies), according to the manufacturer instructions. BiotinylatedEphA5 antigen was used for the in vitro phage and yeast displayselections. Briefly, the scFv naive library cloned into the pDNA5 vectorwas expressed on phage particles. Approximately 10¹² phage particleswere incubated with streptavidin-coated magnetic beads (Dynabeads, LifeTechnologies) saturated with the biotinylated antigen, and 2 rounds ofselection were performed. The selected scFv population was subclonedinto the pDNL6 yeast vector and transformed into yeast.

After induction of the yeast culture, the scFvs were displayed on theyeast cell surface. The cells were incubated with the biotinylatedantigen at 100 nM concentration and the cells displaying the higheraffinity binders were sorted by flow cytometry. After the 2nd round,10,000 antigen-binding yeast cells were sorted and the correspondingdisplayed scFv genes were amplified from the pDNL6 vector by PCR. Theselected scFv genes were cloned back into the phagemid pDAN5 vector anddisplayed on multivalent phage particles (3-5 copies of the scFv weredisplayed per particle).

In vivo scFv antibody selection: The anti-EphA5 scFv phage population(10¹¹ phage particles from the in vitro pre-selected multivalent phagepopulation) was injected intravenously in 3 nude mice bearing xenograftH460 human lung cancer derived tumors. After 3h from the injection, themice were sacrificed and the tumor and control tissues were harvested.The tumor homing phage rescued from the tumor tissue of the 3 mice wereamplified and mixed in equimolar amounts. The in vivo selection wasreiterated for 2 additional cycles. At the end of the 3^(rd) cycle, thescFv genes were amplified by PCR from the pDAN5 plasmid DNA extractedfrom the tissue and prepared for next generation sequencing (NGS). MiSeq(Illumina) paired-end sequencing, combined with a bioinformatic analysisbased on the in house developed AbMining Toolbox software were used toidentify the unique V_(H) domains of the tumor homing scFv genes.

Identification of scFv gene candidates and rescue: The AbMining Toolboxsoftware analysis relies on the recognition of the HCDR3 sequence ofeach scFv gene, a signature element in all antibodies. The sequencedtumor homing clones were identified based on their HCDR3 and rankedaccording to their relative abundance in the tissue. The most abundantclones identified in each round of in vivo selection were chosen ascandidates for further characterization. Once identified, the clones ofinterest were rescued from the selection output by means of an inversePCR-based strategy (D'Angelo et al., 2014, mAbs 6(1): 160-172). Briefly,2 back-to-back primers were designed on the scFv-specific HCDR3 DNAsequence and used for an inverse PCR using the pDAN5 plasmid DNAextracted from the tumor tissue harvested after each selection round.The amplicon obtained are collections of selected HCDR3-specific scFvgenes in the context of the phagemid vector pDAN5. After DNApurification, the amplicons were ligated and transformed into bacteria.Upon sequencing confirmation, the clones were produced as mono- ormulti-valent phage or as Ig-like minibodies (Di Niro et al., 2007, BMCBiotechnology 7:46) into CHO-S cells for further characterization.

Various anti-EphA5 scFv clones were further characterized. Three of themost abundant scFv clones identified were sequenced to determine theirfull length amino acid sequences, presented below. The VL and VH domainsare listed below in Table 1, along with the CDRs.

antiEphA5_E31 scFv amino acid sequence (SEQ ID NO: 33)SYELIQPPSVSVAPGQTARITCGGSNIRSKSVHWY QQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHWVFGGRTKV TVLSGGSTTTSYNVYYTKLSSSGTQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHAAAGDYWGQGTLVTVSSantiEphA5_F31 scFv amino acid sequence (SEQ ID NO: 34)SYELTQPPSVSVAPGKTARITCEGNNIGSKGVHWY QQKPGQAPALVVYDGSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDNSSDHPNYVFGTRT KLTVLSGGSTKTSYNVYYTKLSSSGTQVQLVETGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKG LEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVAAGDYWGQGTLVTVS SantiEphA5_TW3 scFv amino acid sequence (SEQ ID NO: 35)DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGY NYVDWYLQKPGQPPHLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQDPRTFGQ GTKVEIKSGGSTITSYNVYYTKLSSSGTQVQLVESGGGLVQPGGSLRLSCAASGFAFSNYAMSWVRQAPG KGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGAFGGRKGQGTLV TVSSantiEphA5_T22 scFv amino acid sequence (SEQ ID NO: 36)QPVLTQSSSLSASPGASVSLTCTLRSGINVGPYRM YWYQQKPGSPPQYLLSYKSDSDTQQASGVPSRFSGSKDASANAGILLISGLQSEDEADYYCMIWHNNAVV FGGGTKLTVLSGGSTITSYNVYYTKLSSSGTQVQLVQSGTEVKKPGASVKVSCKVSGYSLSELSMHWVRQ APGKGLEWMGSFDPEDGETTYAQRFQGRVTMTEDTSTDTAYMELRSLTSDDTAVYYCAREIWSGYAYFDL WGRGTLVTVSS

TABLE 1 Amino acid sequences and CDRs of scFv clones.Heavy Chain Sequence Light Chain Sequence CDR1 CDR2 CDR3 CDR1 CDR2 CDR3Amino acid sequence Amino acid sequence E31 GFTFSSYA AISGSGGST HAAAGDYGGSNIRSK DDSDRPS QVWDSSSD MS YYADSVKG (SEQ ID SVH (SEQ ID HWV (SEQ ID(SEQ ID NO: 5) (SEQ ID NO: 7) (SEQ ID NO: 3) NO: 4) NO: 6) NO: 8)TQVQLVESGGGLVQPGGSLRLSCAASG SYELIQPPSVSVAPGQTARITCGGSNIFTFSSYAMSWVRQAPGKGLEWVSAISG RSKSVHWYQQKPGQAPVLWYDDSDRPSGGSTYYADSVKGRFTISRDNSKNTLY SGIPERFSGSNSGNTATLTISRVEAGDLQMNSLRAEDTAVYYCATHAAAGDYWG EADYYCQVWDSSSDHWVFGGRTKVTVLQGTLVTVSS (SEQ ID NO : 1) (SEQ ID NO: 2) F31 GFTFSSYA AISGSGGST VAAGDYEGNNIGSK DGSDRPS QVWDNSSD MS (SEQ YYADSVKG (SEQ ID GVH (SEQ (SEQ IDHPNYV ID NO: (SEQ ID NO: 13) ID NO: NO: 15) (SEQ ID 11) NO: 12) 14)NO: 16) TQVQLVETGGGLVQPGGSLRLSCAASG SYELTQPPSVSVAPGKTARITCEGNNIFTFSSYAMSWVRQAPGKGLEWVSAISG GSKGVHWYQQKPGQAPALVVYDGSDRPSGGSTYYADSVKGRFTISRDNSKNTLY SGIPERFSGSNSGNTATLTISRVEAGDLQMNSLRAEDTAVYYCARVAAGDYWGQ EADYYCQVWDNSSDHPNYVFGTRTKLTGTLVTVSS (SEQ ID NO: 9) VL (SEQ ID NO: 10) TW3 GFAFSNYA GISGSGGSTEGAFGGR RSSQSLLH LGSNRAS MQGLQDPR MS (SEQ YYADSVKG K SNGYNYVD (SEQ IDTF (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID NO: 23) ID NO: 19) NO: 20)NO: 21) NO: 22) 24) TQVQLVESGGGLVQPGGSLRLSCAASGDIVMTQTPLSLPVTPGEPASISCRSSQ FAFSNYAMSWVRQAPGKGLEWVSGISGSLLHSNGYNYVDWYLQKPGQPPHLLIY SGGSTYYADSVKGRFTISRDNSKNTLYLGSNRASGVPDRFSGSGSGTDFTLKIS LQMNSLRAEDTAVYYCAREGAFGGRKGRVEAEDVGVYYCMQGLQDPRTFGQGTK QGTLVTVSS (SEQ ID NO : 17)VEIK (SEQ ID NO: 18) T22 GYSLSELS SFDPEDGET EIWSGYA TLRSGINV KSDSDTQMIWHNNAV MH TYAQRFQG YFDL GPYRMY (SEQ ID V (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 31) (SEQ ID NO: 27) NO: 28) NO: 29) NO: 30) NO: 32)TQVQLVQSGTEVKKPGASVKVSCKVSG QPVLTQSSSLSASPGASVSLTCTLRSGYSLSELSMHWVRQAPGKGLEWMGSFDP INVGPYRMYWYQQKPGSPPQYLLSYKSEDGETTYAQRFQGRVTMTEDTSTDTAY DSDTQQASGVPSRFSGSKDASANAGILMELRSLTSDDTAVYYCAREIWSGYAYF LISGLQSEDEADYYCMIWHNNAVVFGG DLWGRGTLVTVSSGTKLTVL (SEQ ID NO: 26) (SEQ ID NO: 25)

A whole cell phage-ELISA was used to demonstrate specific binding of theselected anti-EphA5 scFv antibodies to EphA5 target on a human lungcarcinoma cell line in vitro. Single scFv clones were produced asmonovalent phage and tested for binding on H460 human lung carcinoma(EphA5 positive) and H226 human lung squamous cell carcinoma (EphA5negative) cell lines in vitro. Briefly, cells were seeded on 96-wellplate, blocked and incubated with phage displaying the scFv of interestat different concentrations. After washing, cell surface bound phagewere detected with the antiM13-HRP conjugated mAb (GE).

The results of the binding assay are presented in FIG. 1, and show thatall four of the scFv clones specifically bound to EphA5 positive H460cells.

A whole cell phage-internalization assay was used to characterize theinternalization properties of the selected anti-EphA5 scFv antibodies.Single scFv clones were produced as monovalent phage and tested forbinding and internalization on H460 human lung carcinoma (EphA5positive) and H226 human lung squamous cell carcinoma (EphA5 negative)cell lines in vitro. An irrelevant scFv displayed on phage was used asnegative control. Briefly, the cells were seeded on microscope slides,blocked and incubated with phage for 4 h to allow binding andinternalization. After washing, the surface bound phage were stripped,and the cells were fixed and permeabilized. The phage were detected witha mouse antiM13 mAb (GE) and a Cy3-conjugated antiMouse secondary Ab(Dako). The slides were mounted with VECTASHIELD® anti-fade mountingmedium containing DAPI staining for nuclei and visualized byimmunofluorescence.

The results are presented in FIG. 2, which shows the immunofluorescenceimages of the internalization assay. Two of the tested scFv antibodies(T22 and F31) showed significant internalization properties, with oneshowing very high levels of internalization (T22).

In order to evaluate the therapeutic effectiveness of anti-EphA5antibody-drug conjugates (ADCs), the four anti-EphA5 scFv antibodieswere expressed as minibodies and utilized in a secondary antibody-drugconjugate cell-based cytotoxic assay. More specifically, minibodiescomprising scFv-human Fc fusions were expressed in CHO-S cells (LifeTechnologies) and tested on the EphA5 positive H460 human lung carcinomacell line.

Briefly, supernatant containing minibodies at an unknown concentrationwere added to cells cultured in 96-wells plates. After a 10 minuteincubation on ice, secondary antibody-drug conjugates were added to eachwell at a final concentration of 20 nM. Cell survival was assessed inreal time for 72 h with the automated xCELLigence System (ACEABiosciences). The following secondary antibody-drug conjugates obtainedfrom Moradec LLC, San Diego were evaluated in the cytotoxic assay.

-   1. Fab-aHFc-CL-MMAF: Fab anti-human IgG Fc-MMAF (monomethyl    auristatin F) antibody with cleavable linker.-   2. Fab-aHFc-CL-DMDM: Fab anti-human IgG Fc-duocarmycin antibody with    cleavable linker.-   3. Fab-aHFc-NC-DM1: Fab anti-human IgG Fc-maytansinoid antibody with    non-cleavable linker.-   4. Fab-aHFc-NC-AAMT: Fab anti-human IgG Fc-amanitin antibody with    non-cleavable linker.-   5. Fab-aHFc-NC-AAMF: Fab anti-human IgG Fc-AAMF antibody with    non-cleavable linker.

The results are reported in FIGS. 3A-3B, wherein cell viability wasplotted over time in presence of the minibody in combination with thesecondary ADC or in presence of the secondary ADC alone, and in FIG. 4,wherein cell viability was plotted over concentration. For allminibodies, an enhanced cell killing effect was observed when comparedto the secondary drug conjugates alone. A generally better performancewas observed when the minibodies were used with the DM1 maytansinoidsecondary conjugate, a cytotoxic small molecule which inhibits celldivision by blocking the polymerization of tubulin.

Tumor targeting properties of the minibodies was also analyzed. ProteinG purified minibodies were injected into mice bearing tumors and allowedto circulate for 6 min. After perfusion with PBS and with PFA, tumortissue and controls were harvested, embedded and sectioned. Minibodydetection in tumor and selected control tissues (liver and pancreas) isshown in FIG. 5.

Additionally, the radiosensitization activity of anti-EphA5 clones wasstudied. Surviving fractions of H460 cells exposed to increasing dosesof IR after administration of phage displaying selected anti-EphA5scFvs. A phage library was used as control. Results are shown in FIGS.6A-6D. Two out of the four tested clones (F31 (FIG. 6B) and TW3 (FIG.6D) showed radiosensitizing properties.

Example 2 — Generation of Anti-GRP78 Specific scFv Antibodies

In vitro scFv antibody selection: Recombinant human GRP78 antigen(Abcam) was biotinylated (EZ-Link™ Sulfo-NHS-LC-LC-Biotin, LifeTechnologies), according to the manufacturer instructions. BiotinylatedGRP78 antigen was used for the in vitro phage and yeast displayselections. Briefly, the scFv naive library cloned into the pDNA5 vectorwas expressed on phage particles. Approximately 10¹² phage particleswere incubated with streptavidin-coated magnetic beads (Dynabeads, LifeTechnologies) saturated with the biotinylated antigen, and 2 rounds ofselection were performed. The selected scFv population was subclonedinto the pDNL6 yeast vector and transformed into yeast.

After induction of the yeast culture, the scFvs were displayed on theyeast cell surface. The cells were incubated with the biotinylatedantigen at 100 nM concentration and the cells displaying the higheraffinity binders were sorted by flow cytometry. After the 2nd round,10,000 antigen-binding yeast cells were sorted and the correspondingdisplayed scFv genes were amplified from the pDNL6 vector by PCR. Theselected scFv genes were cloned back into the phagemid pDAN5 vector anddisplayed on multivalent phage particles (3-5 copies of the scFv weredisplayed per particle).

In vivo scFv antibody selection: The anti-GRP78 scFv phage population(10¹¹ phage particles from the in vitro pre-selected multivalent phagepopulation) was injected intravenously in 3 Balb/c mice bearingEF43.fgf4 murine breast cancer derived xenograft tumors. After 3h fromthe injection, the mice were sacrificed and the tumor and controltissues were harvested. The tumor homing phage rescued from the tumortissue of the 3 mice were amplified and mixed in equimolar amounts. Thein vivo selection was reiterated for 2 additional cycles. At the end ofthe 3^(rd) cycle, the scFv genes were amplified by PCR from the pDAN5plasmid DNA extracted from the tissue and prepared for next generationsequencing (NGS). MiSeq (Illumina) paired-end sequencing, combined witha bioinformatic analysis based on the in house developed AbMiningToolbox software were used to identify the unique V_(H) domains of thetumor homing scFv genes.

Identification of scFv gene candidates and rescue: The AbMining Toolboxsoftware analysis relies on the recognition of the HCDR3 sequence ofeach scFv gene, a signature element in all antibodies. The sequencedtumor homing clones were identified based on their HCDR3 and rankedaccording to their relative abundance in the tissue. The most abundantclones identified in each round of in vivo selection were chosen ascandidates for further characterization. Once identified, the clones ofinterest were rescued from the selection output by means of an inversePCR-based strategy (D'Angelo et al., 2014, mAbs 6(1): 160-172). Briefly,2 back-to-back primers were designed on the scFv-specific HCDR3 DNAsequence and used for an inverse PCR using the pDAN5 plasmid DNAextracted from the tumor tissue harvested after each selection round.The amplicon obtained are collections of selected HCDR3-specific scFvgenes in the context of the phagemid vector pDAN5. After DNApurification, the amplicons were ligated and transformed into bacteria.Upon sequencing confirmation, the clones were produced as mono- ormulti-valent phage or as Ig-like minibodies (Di Niro et al., 2007, BMCBiotechnology 7:46) into CHO-S cells for further characterization.

Various anti-GRP78 scFv clones were further characterized. Three of themost abundant scFv clones identified were sequenced to determine theirfull length amino acid sequences, presented below. The VL and VH domainsare listed below in Table 1, along with the CDRs.

antiGRP78_B4 scFv amino acid sequence (SEQ ID NO: 61)SYVLTQPPSVSVAPGKTATITCGGDDIGSKSVHWYQQKPGQAPVLVVYDDGDRPSGIPERFSGSNSGNTATLAISRVEAGDEADYYCQVWDSSSDQYVFGSGTKLTVLSGGSTITSYNVYYTKLSSSGTQVRLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARYSSIDAFEIWGQ GTMVTVSSantiGRP78_D1 scFv amino acid sequence (SEQ ID NO: 62)SYELIQPPSVSVAPGQTARIACGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHPGVFGTGTKLTVLSGGSTITSYNVYYTKLSSSGTQVQLQQSGPGLVEPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSRWYNDYAVSVESRITINPDTSKNQFSLQLNSVTPEDTAVYYCARDPYYYDSSG YYYFDAFGIWGQGTMVTVSSantiGRP78_F6 scFv amino acid sequence (SEQ ID NO: 63)SYELTQPHSVSVAPGQTARITCGGDNIGSKSVHWYQQRPGQAPVLVVYDDSDRPSGIPERFSGSNSENTATLTISGVEAGDEADYYCQVWDSTSHHVVFGGGTKLTVLSGGSTITSYNVYYTKLSSSGTQVQLQQSGPGLVKPPQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARDPYYYDSSGY YYFDAFDIWGQGTMVTVSS

TABLE 1A Amino acid sequences and CDRs of scFv clones.Heavy Chain Sequence Light Chain Sequence CDR1 CDR2 CDR3 CDR1 CDR2 CDR3Amino acid sequence Amino acid sequence B4 GGSISSGG YIYYSGSTY RYSSIDAGGDDIGSK DDGDRPS QVWDSSSD YY YNPSLES FEI SVH (SEQ ID QYV (SEQ (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 43) ID NO: NO: 39) NO: 40) NO: 41) NO: 42)44) QVRLQESGPGLVKPSQTLSLTCTVSGG SYVLTQPPSVSVAPGKTATITCGGDDISISSGGYYWSWIRQHPGKGLEWIGYIY GSKSVHWYQQKPGQAPVLVVYDDGDRPYSGSTYYNPSLESRVTISVDTSKNQFS SGIPERFSGSNSGNTATLAISRVEAGDLKLSSVTAADTAVYYCARYSSIDAFEI EADYYCQVWDSSSDQYVFGSGTKLTVLWGQGTMVTVSS (SEQ ID NO: 37) (SEQ ID NO: 38) D1 GDSVSSNS RTYYRSRWYDPYYYDS GGNNIGSK DDSDRPS QVWDSSSD AA (SEQ NDYAVSVES SGYYYFD SVH (SEQ(SEQ ID HPGV ID NO: (SEQ ID AFGI ID NO: NO: 15) (SEQ ID 47) NO: 48)(SEQ ID 50) NO: 51) NO: 49) QVQLQQSGPGLVEPSQTLSLTCAISGDSYELIQPPSVSVAPGQTARIACGGNNI SVSSNSAAWNWIRQSPSRGLEWLGRTYGSKSVHWYQQKPGQAPVLVVYDDSDRP YRSRWYNDYAVSVESRITINPDTSKNQSGIPERFSGSNSGNTATLTISRVEAGD FSLQLNSVTPEDTAVYYCARDPYYYDSEADYYCQVWDSSSDHPGVFGTGTKLTV SGYYYFDAFGIWGQGTMVTVSS L (SEQ ID NO: 46)(SEQ ID NO: 45) F6 GDSVSSNS RTYYRSKWY DPYYYDS GGDNIGSK DDSDRPS QVWDSTSHAA (SEQ NDYAVSVKS SGYYYFD SVH (SEQ (SEQ ID HVV (SEQ ID NO: (SEQ ID AFDIID NO: NO: 59) ID NO: 55) NO: 56) (SEQ ID 58) 60) NO: 57)QVQLQQSGPGLVKPPQTLSLTCAISGD SYELTQPHSVSVAPGQTARITCGGDNISVSSNSAAWNWIRQSPSRGLEWLGRTY GSKSVHWYQQRPGQAPVLVVYDDSDRPYRSKWYNDYAVSVKSRITINPDTSKNQ SGIPERFSGSNSENTATLTISGVEAGDFSLQLNSVTPEDTAVYYCARDPYYYDS EADYYCQVWDSTSHHVVFGGGTKLTVLSGYYYFDAFDIWGQGTMVTVSS (SEQ (SEQ ID NO: 54) ID NO: 53)

A whole cell phage-internalization assay was used to characterize theinternalization properties of the selected anti-GRP78 scFv antibodies.Single scFv clones were produced as monovalent phage and tested forbinding and internalization on MCF-7 human breast cancer (GRP78positive) cell line in vitro. An irrelevant scFv displayed on phage wasused as negative control. Briefly, the cells were seeded on microscopeslides, blocked and incubated with phage for 4h to allow binding andinternalization. After washing, the surface bound phage were stripped,and the cells were fixed and permeabilized. The phage were detected witha mouse antiM13 mAb (GE) and a Cy3-conjugated antiMouse secondary Ab(Dako). The slides were mounted with VECTASHIELD® anti-fade mountingmedium. The results are presented in FIG. 7, which shows theimmunofluorescence images of the internalization assay. Internalizedphage is shown are red dots inside the cells. Nuclei are stained inblue.

A whole cell phage-ELISA was used to demonstrate specific binding of theselected anti-GRP78 scFv antibodies to GRP78 target on a human breastcarcinoma cell line in vitro. Single scFv clones were produced asmonovalent phage and tested for binding on MCF-7 in vitro. Briefly,cells were seeded on 96-well plate, blocked and incubated with phagedisplaying the scFv of interest at different concentrations. Afterwashing, cell surface bound phage were detected with the antiM13-HRPconjugated mAb (GE). An irrelevant scFv displayed on phage was used asnegative control. The results are shown in FIG. 8. Anti-GRP78 phage bindspecifically to cells expressing surface GRP78. Negative control showsbackground binding.

Cell surface binding of anti-GRP78 minibodies was assessed by flowcytometry on Ef43 murine breast cancer cell line. Briefly, Ef43 cellswere harvested and incubated for 30 min with the fluorescently labeledminibodies (antiGRP78 and isotype control). After extensive washings,the cells were analyzed by flow cytometry. The results are shown in FIG.9. Anti-GRP78 minibodies bind to the surface of breast cancer cells.

In order to evaluate the therapeutic effectiveness of anti-GRP78antibody-drug conjugates (ADCs), the anti-GRP78 scFv antibodies wereexpressed as minibodies and utilized in a secondary antibody-drugconjugate cell-based cytotoxic assay. Briefly, protein G purifiedminibodies were added to cells cultured in 96-wells plates. After a 10minute incubation on ice, secondary antibody-drug conjugates were addedto each well at a final concentration of 20 nM. Cell survival wasassessed in real time for 72 h with the automated xCELLigence System(ACEA Biosciences). The results are shown in FIGS. 10A-10D. Cell killingoccurs in the presence of B4 and D1 minibodies and the drugs AAMT(Alfa-amanitin) and (duocarmycin DM).

Tumor targeting properties of the minibodies was also analyzed. ProteinG purified minibodies were injected into mice bearing tumors and allowedto circulate for 6 min. After perfusion with PBS and with PFA, tumortissue and controls were harvested, embedded and sectioned. Minibodydetection in tumor and selected control tissues (liver and pancreas) isshown in FIG. 8. Minibodies target tumors specifically. Liver andpancreas were used a control organs and showed minimal staining.

Example 3- SPARTA Methodology Experimental Procedures Animals

Female wild-type Balb/c and female Balb/c Nu/Nu mice (HarlanLaboratories) were housed in the animal facility at the University ofNew Mexico Comprehensive Cancer Center (UNMCCC). All animal procedureswere formally reviewed and approved by the Institutional Animal Care andUse Committee (IACUC) at the UNMCCC. Cell Culture

NCI-H460, A549 and NCI-H226 human lung cancer-derived cell lines werepurchased from the American Type Culture Collection (ATCC) andmaintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10%fetal bovine serum (FBS) plus 1% penicillin G/streptomycin SO₄. HumanMCF7 breast cancer-derived cells were purchased from ATCC and maintainedin Eagle's Medium, supplemented with 0.01 mg/mL human recombinantinsulin and 10% FBS. Mouse mammary Ef43.fgf4 cells were maintained inDMEM supplemented with 10% FBS, 5 ng/ml mouse epithelial growth factor(EGF), 1 μg/ml bovine insulin plus 1% penicillin G/streptomycin SO₄.SUM19OPT human inflammatory breast cancer cells were maintained in HamF12 medium, supplemented with 5% FBS, 5 μg/mL insulin, 1 μg/mLhydrocortisone, 10 mM HEPES, and 1% penicillin G/streptomycin SO₄. CHO-Scells were purchased from Life Technologies and maintained in serum-freeFreeStyle™ CHO Expression Medium. Ef43.fgf4 GRP78-knockdown cells wereobtained by lentivirus infection, stably transfected and maintained inselection media containing antibiotics. The Quantum™ Simply Cellular®Microspheres (Bangs Laboratories) were used to quantitate the number oftargeted molecules on the surface of cells.

Phage-And Yeast-Display Antibody Screening In Vitro

scFv antibody clones were isolated by integrating phage- andyeast-display methodologies as described (Ferrara et al., 2015).Briefly, a naïve human phage antibody library (Sblattero and Bradbury,2000) was used in two rounds of screening in vitro either on recombinanthuman EphA5 (R&D Systems) or GRP78 (Abcam). The binding pools of scFvclones were subcloned into a yeast display vector as described (Ferraraet al., 2012), and the yeast mini-libraries were further enriched fortarget-specific binders applying two rounds of sorting by using standardflow cytometry protocols (FACSAria, Becton Dickinson) as described(Boder and Wittrup, 1998).

Experimental Tumor Xenograft And Isogenic Models

Tumor targeting properties of the minibodies was also analyzed. ProteinG puri Mouse mammary Ef43.fgf4 isogenic tumor cells and SUM19OPT humaninflammatory breast cancer cells were collected at 70% confluency andadministered subcutaneously (s.c.) in the mammary fat pad of eitherimmunocompetent female Balb/c mice (Ef43.fgf4), or female Balb/c nudemice (SUM190PT) as indicated. SUM190PT cells were administered s.c. in1:1 (vol/vol) with Matrigel™ (Corning) as described (Dobroff et al.,2016). NCI-H460 human lung cancer cells were administered s.c. in theright flanks of Balb/c female nude mice. After approximately 10 d,tumors reached —200-300 mm³ and were separated into size-matchedtumor-bearing mouse cohorts for experimentation.

Phage-Display In Vivo

In vivo phage selections were performed as described (Kolonin et al.,2006; Pasqualini and Ruoslahti, 1996; Rajotte et al., 1998; Staquiciniet al., 2011b). Animals received 10¹⁰ phage particles i.v. and tumorsand negative control organs were collected after 3 h of systemiccirculation. Tumor-homing phage were retrieved by PCR amplification, andfull-length scFv products (˜800 bp) were re-cloned to producemultivalent functional phage particles for subsequent rounds ofselection. Phage quantification was performed by quantitative phage PCR(Dias-Neto et al., 2009) and host bacterial infection as described(Kolonin et al., 2006; Rajotte et al., 1998; Staquicini et al., 2011a;Staquicini et al., 2011b).

Phage Binding Assays And Enzyme-Linked Immunosorbent Assay (ELISA)

Serial dilutions of individual phage particles in phosphate-bufferedsaline (PBS) containing 2% non-fat milk were placed in microtiter wellspreviously coated with 0.5 μg of either EphA5 or GRP78, as indicated.After extensive washes, remaining bound phage particles were detectedwith an anti-M13 peroxidase-conjugated mouse monoclonal antibody(GE-Amersham Biosciences).

Phage binding to the surface of cells was tested by whole-cell ELISA.Briefly, exponentially growing cells were fixed in 96-wellmicrotitration plates (Nunc) at 3×10⁵ cells/well and exposed to serialdilutions of either targeted or control phage particles for 2 h at roomtemperature (RT). Wells were extensively washed with PBS containing 0.1%Tween-20, and bound particles were detected with an anti-M13peroxidase-conjugated mouse monoclonal antibody. A non-targeted helperphage (termed M13KO7 (Ferrara et al., 2012)) and unrelated scFv servedas negative controls, as indicated.

Cell Internalization Assay

Cell internalization assays were performed as described (Arap et al.,2004). In brief, cells plated in eight-chamber slides were blocked withDMEM containing 2% FBS for 1 h at RT, and incubated with 10⁹ TU ofphage. After 2 h of incubation at 37° C., cell membrane-bound phage wereremoved by washes with 20 mM glycine (pH 2.3), and fixed with PBScontaining 4% paraformaldehyde (PFA). Fixed cells were permeabilizedwith PBS containing 0.1% Triton X-100, blocked with PBS containing 1%bovine serum albumin (BSA), and incubated with a mouse anti-M13 phagemonoclonal antibody for 1 h at RT. After incubation with a rabbitanti-mouse IgG Cy3-conjugated secondary antibody (JacksonImmunoresearch), cells were washed with PBS, and re-fixed in PBScontaining 4% PFA. Internalized phage particles were visualized with astandard fluorescence microscope (Nikon Ti-E Inverted Microscope,Nikon).

NGS And Data Analysis

Sample preparation for NGS was performed as described (D'Angelo et al.,2014a). Briefly, plasmid DNA recovered from the selection outputs wasamplified with a specific set of primers designed for the MiSeqpaired-end sequencing of the scFv VH domains. The amplicons weresequenced with the MiSeqV2 kit for 500 cycles. Sequencing results wereanalyzed with the aid of the AbMining toolbox software package withdefault settings for quality filtering (D'Angelo et al., 2014a; D'Angeloet al., 2014b). The identified HCDR3s were clustered at Hamming distance1 and analyzed further in MS Excel (D'Angelo et al., 2014a; D'Angelo etal., 2014b).

Scfv-Fc Production

Monoclonal scFv antibody genes were subcloned either into the pHygrovector for scFv-Fc expression in CHO cells, or into the yeast expressionvector pDNL9 for expression into YVH10 S. cerevisiae yeast cells asindicated (Ferrara et al., 2012), both containing a human Fc sequence(Ferrara et al., 2015). All cloning steps were monitored and verified byDNA restriction enzyme digestion analysis and sequencing. scFv-Fcfusions were purified from the culture supernatant by affinitypurification on Protein-G agarose (Roche). ELISA with the correspondingtarget was performed to test binding specifity of individually purifiedscFv-Fc.

In Vivo Scfv-Fc Targeting

Tumor homing was assessed by i.v. administration of the scFv-Fc poolinto tumor-bearing mice. Two doses of a 2.5 μM solution (100 and 200 μL)were administered i.v. into the tail vein of anesthesized tumor-bearingmice. The scFv-Fcs were allowed to circulate for 6 min, prior tofull-body cardiac perfusion with PBS. Immunofluorescence staining wasperformed on tissue sections with a Cy3-conjugated goat anti-human FcγIgG (Jackson Immunoresearch) and DAPI for nuclei staining. Images wereacquired with an Nikon Ti-E Inverted fluorescence microscope.

In Vitro Cytotoxicity Assays

Cell killing activity was measured in real-time with the Xcelligencesystem (ACEA Biosciences). Freshly split tumor cells (25,000 cells/well)were cultured overnight (ON) in a 96-well electronic microtiter plate(E-plate® 96) (ACEA Biosciences) in 100 μL of complete culture medium.After 24 h, increasing concentrations of the primary monoclonal scFv-Fcswere added to each microwell followed by addition of 20 nM of secondaryADC reagents (Moradec LLC) linked to monomethyl auristatin F (MMAF)(Fab-αHFc-CL-MMAF), duocarmycin (DMDM) (Fab-αHFc-CL-DMDM), emtansine(DM1) (Fab-αHFc-NC-DM1), or amanitin (AAMT) (Fab-αHFc-NC-AAMT). Cellindex was measured every 30 min for 96 h. Controls included primaryscFv-Fc alone, drug-conjugated secondary alone, and non-treated cells asindicated.

RESULTS Serial In Vitro Screening and In Vivo Selection for theDiscovery of Human Recombinant Monoclonal Antibodies

A schematic representation of SPARTA is depicted in FIGS. 12A-12C. Wefirst carried out an in vitro unbiased library screening againstimmobilized human recombinant EphA5 and GRP78 from a large human naivephage-displayed single chain variable fragment (scFv) library (Sblatteroand Bradbury, 2000). After two rounds of library screening in vitro,diversity was reduced five orders of magnitude (from ˜10¹¹ unique scFvsequences to ˜10⁶). The total phage pool output was subsequently clonedinto a yeast-display system. This maneuver allows precise fluorescenceactivated cell sorting to restrict reactivity to those clonesrecognizing the target (Ferrara et al., 2015). After two additionalrounds of yeast cell sorting, a diverse highly enriched antibodypopulation was obtained for each target (FIGS. 12B and 12C). Antibodyclones bound specifically to the corresponding antigens, in both yeast-and phage-display contexts (FIGS. 12B and 12C). The anti-EphA5 antibodypools were negatively selected to minimize or eliminate anti-EphA5antibodies that would also recognize orthologous ephrin-family members(namely EphA3, 4, 6, and 7) (FIG. 16). Next-generation sequencing (NGS)plus AbMining Toolbox analysis (D'Angelo et al., 2014a) confirmed highlydiverse antibody populations, as assessed by the sizable number ofindividual heavy chain complementarity-determining region 3 (HCDR3)variants obtained for EphA5 (n=207) and GRP78 (n=125). The relativelyhigh polyclonality observed underscores the power of the combinedphage/yeast display approach to select diverse specific antibodies.

Based on the current understanding of the importance of multivalency inin vivo peptide phage-display originally developed by our group (Arap etal., 2002; Arap et al., 1998; Barnhart et al., 2011; Christianson etal., 2007; Dias-Neto et al., 2009; Hajitou et al., 2006; Kolonin et al.,2001; Kolonin et al., 2004; Kolonin et al., 2006; Ozawa et al., 2008;Pasqualini and Arap, 2002; Pasqualini and Ruoslahti, 1996; Rajotte etal., 1998; Staquicini et al., 2011a; Staquicini et al., 2011b), wegenerated multivalent antibody phage displaying anti-EphA5 andanti-GRP78 scFv populations using a helper plasmid technology (Chasteenet al., 2006). As opposed to traditional helper phage-mediatedmonovalent antibody display, helper plasmids provide all the requiredviral packaging functions but without helper phage contamination in amultivalent form, thereby reducing background binding during in vivoselections, and increasing specific binding of targeted phage throughimproved avidity (Chasteen et al., 2006). While it has proved somewhatchallenging to use helper plasmids with large naïve antibody libraries,applications to peptide libraries and small targeted antibody librariesby contrast have been much more successful (Phipps et al., 2016). Therecombinant scFv genes previously sorted by yeast-display were clonedback into the phagemid display vector pDAN5 (Sblattero and Bradbury,2000), as a polyclonal pool, and transformed into E.coli carrying theM13cp-dg3 helper plasmid (Chasteen et al., 2006; Phipps et al., 2016).The polyclonal multivalent phage pools were then administeredintravenously (i.v.) into breast tumor- or lung tumor-bearing mice.Tumor xenografts (NCI-H460 human lung cancer cells for EphA5) orisogenic tumors (Ef43.fgf4 murine breast cancer cells for GRP78), andcontrol organs were collected after 3 hours to enrich for phage clonesthat exclusively localized to tumors in vivo (FIGS. 12D and 12E). Therelative number of phage particles in tumor and control tissue sampleswas assessed by quantitative real-time PCR. Amplified scFv genesisolated from the tumors were recloned back into the originalphage-display vector and used for serial rounds of selection in vivo.After three rounds, specific targeting of EphA5 (FIG. 12D) andGRP78-expressing tumors (FIG. 12E), compared to several negative controlorgans, was observed. Two non-mutually exclusive approaches were used toidentify lead monoclonal antibody candidates: for GRP78-targetingantibodies, phage clones from the final round of in vivo selection wereevaluated for binding to the cognate recombinant antigen. ForEphA5-targeting antibodies, the enrichment of the tumor-homing cloneswas assessed by NGS and the top-ranking clones determined by frequencywere chosen as the lead monoclonal antibody candidates. In both cases,monoclonal antibodies were verified by DNA sequencing and restrictionenzyme digestion to be confirmed full-length scFvs, with norearrangements, stop codons or frameshifts.

Validation of Tumor-Homing In Vivo

A total of four scFv clones against EphA5 (termed E1-4) and threeagainst GRP78 (termed G1-3) were selected for functional studies (FIG.22). Monoclonal antibodies were again displayed multivalently on phage,and tested individually for their ability to home and localize to tumorsin vivo after i.v. administration. Phage displaying a scFv antibodyagainst M2 (Gabbard et al., 2009), a conserved influenza virus protein,served as a standard negative control. Individual phage clones wereadministered i.v. in tumor-bearing mice and tumor tissue and controlorgans were recovered after 3 hours (FIGS. 13 and 14). Relativequantification of phage particles in tissue samples revealed markedaccumulation of EphA5-binding phage to EphA5-expressing tumors (FIG.13). On the other hand, GRP78-binding phage homed and localized toGRP78-expressing tumors (FIG. 14), compared to several negative controlorgans (shown are brain, muscle, and pancreas). No tumor homing wasdetected by control phage (insertless phage and phage displaying a scFvantibody against M2) in either experimental tumor model.

Assessment of Functional Binding Specificity

Having validated tumor-homing in vivo, we next evaluated bindingspecificity to recombinant proteins and endogenous corresponding targetsexpressed on the tumor cell surface. All individual monoclonalantibodies in multivalent phage-display format bound to their respectiveantigens, as measured by recombinant protein-based ELISA (FIGS. 15A-15B)and cell-based ELISA. Cells expressing surface EphA5 or GRP78 alsoshowed receptor-mediated cell internalization of targeted phage clones.Negative controls included an insertless phage, phage displaying anunrelated scFv, and control cells. In order to test the antibodies asfunctional proteins, the scFvs were cloned into expression vectors inthe scFv-human-Fc fusion format and were produced from either ChineseHamster Ovary (CHO) cell culture supernatants, or the S. cerevisiaeexpression system, as indicated. Anti-GRP78 scFv-Fcs recognized humanGRP78 on ELISA and flow cytometry (FIG. 17). Similarly, anti-EphA5scFv-Fcs bound to immobilized recombinant EphA5, and endogenous EphA5expressed on the cell surface (FIGS. 18A-18B). Moreover, when evaluatedin vivo, the scFv-Fcs were able to infiltrate and localize to tumorsafter a 6 minutes circulation time, as assessed by immunofluorescence(FIG. 19). Taken together, these results indicate that the combinationof in vitro screenings on recombinant cell surface receptors followed byin vivo selection of receptor-specific antibodies yields antibody cloneswith favorable on-target biodistribution.

Monoclonal Antibodies have Specific Cytotoxicity Against Lung Cancer andBreast Cancer Cells

ADCs were tested by using drug-conjugated secondary reagents recognizingthe Fc region to deliver cytotoxic drugs. Monoclonal antibody candidateswere prioritized based on binding and receptor-mediated internalizationin target-expressing cells, and a cytotoxicity assay was used todetermine the potency of the lead ADC candidates in a cell-based assay.Binding specificity and antigen-dependent efficacy were assessed withtumor cell lines expressing variable levels of the targets on theirsurfaces (FIG. 20). Negative controls included cells exposed to theprimary scFv-Fc alone, cell lines depleted of EphA5 and GRP78, anon-specific primary scFv-Fc, and drug-conjugated secondary antibodyalone.

Tumor cells were exposed to increasing concentrations of the leadscFv-Fcs, followed by secondary reagents conjugated to a representativepanel of cytotoxic drugs: alpha-amanitin (AAMT), monomethyl auristatin F(MMAF), duocarmycin DM (DMDM), and emtansine (DM1). EphA5-expressingcells showed sensitivity to the scFv-Fc E4, when combined with allcytotoxic drugs. In particular, a clear concentration-dependent responseat a low nanomolar range was observed for both AAMT and DMDM, supportingthe functional retention of both receptor-binding specificity and cellinternalization attributes. Similarly, the anti-GRP78 scFv-Fc G1 wasfound most efficient at inducing cell death, although at a highernanomolar range. Control tumor cells were not affected under the sameexperimental conditions. Incubation with an irrelevant negative controlscFv-Fc or drug-conjugated secondary antibody alone did not result indetectable tumor cell death. A complete panel of ADC experiments isdepicted for reference (FIGS. 21A-21D).

DISCUSSION

A long-term goal of monoclonal antibody-based drug development is toproduce recombinant human monoclonal antibodies with the lowest expectedrisk of immunogenicity. This is usually carried out by immunization oftransgenic mice (Bruggemann et al., 2015) for production of humanantibodies, humanization (Jones et al., 1986; Morrison et al., 1984;Queen et al., 1989; Riechmann et al., 1988) of murine antibodies(Gerhard et al., 1978; Kohler and Milstein, 1975; Koprowski et al.,1977; Pasqualini and Arap, 2004), or in vitro display methods,particularly phage (Marks et al., 1991) and/or yeast-display (Boder andWittrup, 1997). The latter method does not depend on immunization, andmay be used to identify human monoclonal antibodies directly from largenaïve human libraries (Sblattero and Bradbury, 2000). The inventors haverecently combined these two complementary antibody display platformsinto a selection strategy that provides two key potential advantagesover either platform alone: the ability to screen a vast number ofclones in a single experiment, and to tailor the selection for precisefunctional attributes (Ferrara et al., 2012).

In the present application, the inventors introduce the SPARTAmethodology, which serially integrates a two-step strategy based on invitro screening and in vivo selection, to yield a robust monoclonalantibody discovery pipeline. Technical improvements include therecloning of sorted yeast-display antibodies back into our phage displayvector (Sblattero and Bradbury, 2000), displaying them in a multivalentformat (Chasteen et al., 2006), administering them systemically intotumor-bearing mice and isolating those that homed to tumors. The invitro screening selections resulted in a range of hundreds of humanrecombinant antibodies against EphA5 and GRP78, while the in vivofunctional selection identified those antibodies from the polyclonalpool able to recognize cell surface-associated targets within their invivo context.

The use of two non-mutually exclusive approaches served to reduce thenumber of candidate targeted monoclonal antibodies into a practicalbiological number that could then be functionally evaluated. Anotherrational for our choice was to show the robustness and versatility ofSPARTA and, to have an initial glimpse of whether or not one of thesetwo empiric strategies might be either qualitatively superior andquantitatively cost-effective. First, a stochastic approach was appliedto the GRP78-targeting selection, where a random screening of individualclones (n=45) led to the identification of three different scFvs. In thecase of EphA5 we carried out deep NGS before and after each step ofselection in vivo, allowing us to identify EphA5-binding clones thatwere highly enriched in the tumor. With either of these two independentapproaches, we were able to isolate several different antibodies foreach target that homed specifically and localized at the correspondingtumor models. These candidates were further investigated asphage-displayed monoclonal antibodies individually, and were shown to:(i) bind to their respective antigens; (ii) bind tumor lines expressingthe antigens on their cell surface membrane; and (iii) undergoreceptor-mediated internalization into target-expressing tumor cells.Together, these data provide strong evidence that SPARTA producestargeted monoclonal antibodies with translation potential since theefficacy of monoclonal antibody-based therapy relies on their selectiveuptake by cancer cells. When produced in the scFv-Fc format, antibodyfusions retained all the binding functions of the original scFvs andtumor cell death was uniquevocally demonstrated with ADCs (FIGS.21A-21D).

In summary, this study establishes SPARTA as a robust methodology forprompt identification of tumor-targeting human recombinant monoclonalantibodies with high specificity against established cell surfaceantigens. The results presented herein show that SPARTA may well becomea methodology-of-choice to develop therapeutic antibodies from largehuman monoclonal antibody libraries.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, as well as other references cited in thepresent application, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. An isolated monoclonal antibody, wherein theantibody specifically binds to GRP78 and comprises: a heavy chainvariable region (VH) comprising a VH CDR1 having the amino acid sequenceset forth in SEQ ID NO: 39, a VH CDR2 having the amino acid sequence setforth in SEQ ID NO: 40, and a VH CDR3 having the amino acid sequence setforth in SEQ ID NO: 41; and a light chain variable region (VL)comprising a VL CDR1 having the amino acid sequence set forth in SEQ IDNO: 42, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO:43, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO:44.
 2. The antibody of claim 1, wherein the antibody comprises a VHhaving an amino acid sequence that is at least 90% identical to SEQ IDNO: 37 and a VL having an amino acid sequence that is at least 90%identical to SEQ ID NO:
 38. 3. The antibody of claim 1, wherein theantibody comprises a VH having an amino acid sequence that is at least95% identical to SEQ ID NO: 37 and a VL having an amino acid sequencethat is at least 95% identical to SEQ ID NO:
 38. 4. The antibody ofclaim 1, wherein the antibody comprises the VH having the amino acidsequence set forth in SEQ ID NO: 37 and the VL having the amino acidsequence set forth in SEQ ID NO:
 38. 5. The antibody of claim 1, whereinthe antibody is a recombinant antibody.
 6. The antibody of claim 1,wherein the antibody is an IgG, IgM, IgA, or an antigen binding fragmentthereof.
 7. The antibody of claim 1, wherein the antibody is a Fab′, aF(ab′)2, a F(ab′)3, a monovalent scFv, a bivalent scFv, or a singledomain antibody.
 8. The antibody of claim 1, wherein the antibody is afull length antibody.
 9. The antibody of claim 1, wherein the antibodyis a human, humanized antibody, or de-immunized antibody.
 10. Animmunoconjugate comprising the antibody of claim 1 conjugated to atleast one effector moiety selected from an imaging agent, achemotherapeutic agent, a toxin, and a radionuclide.
 11. Theimmunoconjugate of claim 10, wherein the effector moiety is a toxin. 12.The immunoconjugate of claim 11, wherein the toxin is auristatin ormonomethyl auristatin E (MMAE).
 13. The immunoconjugate of claim 12,wherein the toxin is MMAE.
 14. A composition comprising the antibody ofclaim 1 and at least one pharmaceutically acceptable carrier.
 15. Acomposition comprising the immunoconjugate of claim 10 and at least onepharmaceutically acceptable carrier.
 16. A composition comprising theimmunoconjugate of claim 12 and at least one pharmaceutically acceptablecarrier.
 17. A composition comprising the immunoconjugate of claim 13and at least one pharmaceutically acceptable carrier.
 18. Apolynucleotide molecule comprising a nucleic acid sequence encoding theantibody of claim
 1. 19. A method of treating or ameliorating a cancerin a subject, the method comprising administering to the subject aneffective amount of the antibody of claim
 1. 20. A method of treating orameliorating a cancer in a subject, the method comprising administeringto the subject an effective amount of the immunoconjugate of claim 10.21. A method of detecting a cancer in a subject, comprising testing forthe presence of elevated GRP78 in a sample from the subject relative toa control, wherein the testing comprises contacting the sample with theantibody of claim
 1. 22. A method of detecting GRP78, neutralizingGRP78, or counteracting the effects of GRP78, the method comprisingcontacting the GRP78 with the antibody of claim
 1. 23. The method ofclaim 22, wherein the GRP78 is in a cultured cell.
 24. The method ofclaim 22, wherein the GRP78 is on a surface of a cell.
 25. The method ofclaim 22, wherein the GRP78 is in a subject.
 26. The method of claim 25,wherein the GRP78 is in or on a surface of a cancer cell in the subject.27. The method of claim 26, wherein the subject is a human.